Sample indexing for single cells

Abstract

To provide systems, methods, compositions, and kits for sample identification.SOLUTION: Disclosed herein are systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcodes and lengthened using daisy-chaining primers.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 515,285, filed June 5, 2017; U.S. Provisional Patent Application No. 62 / 532,905, filed July 14, 2017; U.S. Provisional Patent Application No. 62 / 532,949, filed July 14, 2017; U.S. Provisional Patent Application No. 62 / 532,971, filed July 14, 2017; U.S. Provisional Patent Application No. 62 / 554,425, filed September 5, 2017; U.S. Provisional Patent Application No. 62 / 578,957, filed October 30, 2017; and U.S. Provisional Patent Application No. 62 / 645,703, filed March 20, 2018. The contents of each of these related applications are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled Sequence_Listing_BDCRI_033A.txt, created on March 20, 2018, and having a size of 3,181 bytes. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present disclosure relates generally to the field of molecular biology, for example, using molecular barcoding to identify cells in various samples and detect interactions between cellular components. [Background technology]

[0004] Current technology allows for the measurement of gene expression in single cells in a massively parallel manner (e.g., in over 10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells, with each cell co-localized in a compartment with a barcoded reagent bead. Gene expression can affect protein expression. Protein-protein interactions can affect gene expression and protein expression. Systems and methods are needed that can quantitatively analyze protein expression, simultaneously measure protein expression and gene expression in cells, and determine protein-protein interactions in cells. Summary of the Invention

[0005] Disclosed herein includes methods for sample identification. In some embodiments, the methods include contacting one or more cells from each of a plurality of samples with a sample indexing composition of a plurality of sample indexing compositions, wherein the one or more cells comprise one or more antigen targets, at least one sample indexing composition of the plurality of sample indexing compositions comprises two or more antigen binding reagents (e.g., a protein binding reagent and an antibody), each of the two or more antigen binding reagents being associated with a sample indexing oligonucleotide, at least one of the two or more antigen binding reagents being capable of specifically binding to at least one of the one or more antigen targets, the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprising different sequences. barcoding sample-indexing oligonucleotides using the plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides; obtaining sequencing data for the plurality of barcoded sample-indexing oligonucleotides; and identifying a sample origin of at least one cell of one or more cells based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides (e.g., identifying a sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded sample-indexing oligonucleotide). The method may, for example, include removing unbound sample-indexing compositions from the plurality of sample-indexing compositions.

[0006] In some embodiments, the sample indexing sequences are 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, or at least 128 nucleotides in length. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. The sample indexing sequences of at least 100 or 1000 sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences.

[0007] In some embodiments, the antigen-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the antigen-binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the antigen-binding reagent. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0008] In some embodiments, at least one sample of the plurality of samples comprises a single cell. At least one of the one or more antigen targets may be on a cell surface or inside a cell. One sample of the plurality of samples may comprise a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0009] In some embodiments, removing unbound sample-indexing composition comprises washing one or more cells from each of the plurality of samples with a wash buffer. The method may comprise lysing one or more cells from each of the plurality of samples. The sample-indexing oligonucleotide may be configured to be detachable or non-detachable from the antigen-binding reagent. The method may comprise detaching the sample-indexing oligonucleotide from the antigen-binding reagent. The detaching of the sample-indexing oligonucleotide may comprise detaching the sample-indexing oligonucleotide from the antigen-binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0010] In some embodiments, the sample indexing oligonucleotide is not homologous to the genomic sequence of cells of the plurality of samples. The sample indexing oligonucleotide may comprise a molecular label sequence, a poly(A) region, or a combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(A) tail. The sample indexing oligonucleotide may comprise a molecular label.

[0011] In some embodiments, the antigen target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The antigen target may be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The antigen target may be or include a lipid, a carbohydrate, or any combination thereof. The antigen target may be selected from a group comprising 10 to 100 different antigen targets. The antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having the same sequence. The antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences. The sample-indexing composition of the plurality of sample-indexing compositions may include a second antigen-binding reagent that is not bound to the sample-indexing oligonucleotide. The antigen-binding reagent and the second antigen-binding reagent may be identical.

[0012] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence. The molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences. The barcodes may comprise a cell beacon, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0013] In some embodiments, the plurality of barcodes are associated with a particle. At least one barcode of the plurality of barcodes can be immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof. The particle can be degradable. The particle can be a bead. The bead can be selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise at least 10,000 barcodes. In some embodiments, the barcodes of the particles may comprise molecular beacon sequences selected from at least 1,000 or 10,000 different molecular beacon sequences. The molecular beacon sequences of the barcodes may comprise random sequences.

[0014] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0015] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data may include sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

[0016] In some embodiments, identifying the sample origin of the at least one cell may include identifying the sample origin of the plurality of barcoded targets based on the sample-indexed sequence of at least one barcoded sample-indexed oligonucleotide. Barcoding the sample-indexed oligonucleotides with the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides may include probabilistically barcoding the sample-indexed oligonucleotides with the plurality of probabilistic barcodes to generate the plurality of probabilistic barcoded sample-indexed oligonucleotides.

[0017] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, each of the plurality of barcodes comprising a cell label, and at least two of the plurality of barcodes comprising an identical cell label sequence; and acquiring sequencing data for the barcoded targets. Barcoding a plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. Prior to acquiring sequencing data for the plurality of barcoded targets, the method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding a plurality of cellular targets using a plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding a plurality of cellular targets using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0018] In some embodiments, each of the plurality of sample indexing compositions comprises an antigen-binding reagent. The sample indexing sequences of the sample indexing oligonucleotides associated with two or more antigen-binding reagents may be identical. The sample indexing sequences of the sample indexing oligonucleotides associated with two or more antigen-binding reagents may comprise different sequences. Each of the plurality of sample indexing compositions may comprise two or more antigen-binding reagents.

[0019] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with one sample indexing composition of a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more cellular component targets, at least one sample indexing composition of the plurality of sample indexing compositions comprises two or more cellular component binding reagents (e.g., antigen binding reagents or antibodies), each of the two or more cellular component binding reagents associated with a sample indexing oligonucleotide, at least one of the two or more cellular component binding reagents capable of specifically binding to at least one of the one or more cellular component targets, and the sample indexing oligonucleotide is associated with a sample indexing oligonucleotide. The method may include the steps of: barcoding the sample-indexing oligonucleotides using the plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides; obtaining sequencing data for the plurality of barcoded sample-indexing oligonucleotides; and identifying a sample origin of at least one cell of the one or more cells based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides. The method may include removing unbound sample-indexing compositions from the plurality of sample-indexing compositions.

[0020] In some embodiments, the sample indexing sequences are 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, or at least 128 nucleotides in length. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions can comprise different sequences. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions can comprise different sequences. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0021] In some embodiments, the cellular component binding reagent comprises a cell surface binding reagent, an antibody, a tetramer, an aptamer, a protein scaffold, an integrin, or a combination thereof. The sample-indexing oligonucleotide may be attached to the cellular component binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the cellular component binding reagent. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0022] In some embodiments, at least one sample of the plurality of samples comprises a single cell. At least one of the one or more cellular component targets may be expressed on a cell surface. One sample of the plurality of samples may comprise a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0023] In some embodiments, removing unbound sample-indexing composition comprises washing one or more cells from each of the plurality of samples with a wash buffer. The method may comprise lysing one or more cells from each of the plurality of samples. The sample-indexing oligonucleotide may be configured to be detachable or non-detachable from the cellular component binding reagent. The method may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent. The detaching the sample-indexing oligonucleotide may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0024] In some embodiments, the sample indexing oligonucleotide is not homologous to the genomic sequence of cells of the plurality of samples. The sample indexing oligonucleotide may comprise a molecular label sequence, a poly(A) region, or a combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(A) tail. The sample indexing oligonucleotide may comprise a molecular label.

[0025] In some embodiments, the cellular component target is or includes a carbohydrate, lipid, protein, extracellular protein, cell surface protein, cell marker, B cell receptor, T cell receptor, major histocompatibility complex, tumor antigen, receptor, integrin, intracellular protein, or any combination thereof. The cellular component target can be selected from a group including 10 to 100 different cellular component targets. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having the same sequence. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having different sample indexing sequences. The sample indexing composition of the multiple sample indexing compositions can include a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent can be the same.

[0026] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence. The molecular beacon sequences of at least two barcodes of the plurality of barcodes may comprise different molecular beacon sequences. The barcode may comprise a cell beacon, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0027] In some embodiments, the plurality of barcodes are encapsulated in particles. The particles may be beads. At least one barcode of the plurality of barcodes may be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof. The particles may be degradable. The beads may be selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise at least 10,000 barcodes. In some embodiments, the barcodes of the particles may comprise molecular beacon sequences selected from at least 1,000 or 10,000 different molecular beacon sequences. The molecular beacon sequences of the barcodes may comprise random sequences.

[0028] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0029] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data may include sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

[0030] In some embodiments, identifying the sample origin of the at least one cell may include identifying the sample origin of the plurality of barcoded targets based on the sample-indexed sequence of at least one barcoded sample-indexed oligonucleotide. Barcoding the sample-indexed oligonucleotides with the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides may include probabilistically barcoding the sample-indexed oligonucleotides with the plurality of probabilistic barcodes to generate the plurality of probabilistic barcoded sample-indexed oligonucleotides.

[0031] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, each of the plurality of barcodes comprising a cell label, and at least two of the plurality of barcodes comprising an identical cell label sequence; and acquiring sequencing data for the barcoded targets. Barcoding a plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. The method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to acquiring sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding a plurality of cellular targets using a plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding a plurality of cellular targets using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0032] In some embodiments, each of the plurality of sample-indexing compositions comprises a cellular component binding reagent. The sample-indexing sequences of the sample-indexing oligonucleotides associated with two or more cellular component binding reagents may be identical. The sample-indexing sequences of the sample-indexing oligonucleotides associated with two or more cellular component binding reagents may comprise different sequences. Each of the plurality of sample-indexing compositions may comprise two or more cellular component binding reagents.

[0033] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with one sample indexing composition from a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more cellular component targets, and at least one sample indexing composition from the plurality of sample indexing compositions comprises two or more cellular component binding reagents, each of the two or more cellular component binding reagents being associated with a sample indexing oligonucleotide, at least one of the two or more cellular component binding reagents being capable of specifically binding to at least one of the one or more cellular component targets, the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions from the plurality of sample indexing compositions comprising different sequences; and identifying the sample origin of at least one of the one or more cells based on the sample indexing sequence of the at least one sample indexing oligonucleotide from the plurality of sample indexing compositions. The method may, for example, include removing unbound sample indexing composition from the plurality of sample indexing compositions.

[0034] In some embodiments, the sample indexing sequences are 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, or at least 128 nucleotides in length. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. The sample indexing sequences of at least 100 or 1000 sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences.

[0035] In some embodiments, the antigen-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the antigen-binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the antigen-binding reagent. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0036] In some embodiments, at least one sample of the plurality of samples comprises a single cell. At least one of the one or more antigen targets may be expressed on a cell surface. One sample of the plurality of samples may comprise a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise mammalian samples, bacterial samples, viral samples, yeast samples, fungal samples, or any combination thereof.

[0037] In some embodiments, removing unbound sample-indexing composition comprises washing one or more cells from each of the plurality of samples with a wash buffer. The method may comprise lysing one or more cells from each of the plurality of samples. The sample-indexing oligonucleotide may be configured to be detachable or non-detachable from the antigen-binding reagent. The method may comprise detaching the sample-indexing oligonucleotide from the antigen-binding reagent. The detaching of the sample-indexing oligonucleotide may comprise detaching the sample-indexing oligonucleotide from the antigen-binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0038] In some embodiments, the sample indexing oligonucleotide is not homologous to the genomic sequence of cells of the plurality of samples. The sample indexing oligonucleotide may comprise a molecular label sequence, a poly(A) region, or a combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(A) tail. The sample indexing oligonucleotide may comprise a molecular label.

[0039] In some embodiments, the antigen target is or includes a carbohydrate, lipid, protein, extracellular protein, cell surface protein, cell marker, B cell receptor, T cell receptor, major histocompatibility complex, tumor antigen, receptor, intracellular protein, or any combination thereof. The antigen target may be selected from a group comprising 10 to 100 different antigen targets. An antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having the same sequence. An antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences. A sample-indexing composition of the plurality of sample-indexing compositions may include a second antigen-binding reagent that is not bound to a sample-indexing oligonucleotide. The antigen-binding reagent and the second antigen-binding reagent may be identical.

[0040] In some embodiments, identifying the sample origin of the at least one cell includes barcoding sample-indexing oligonucleotides of a plurality of sample-indexing compositions with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides; obtaining sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides.

[0041] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence. The molecular beacon sequences of at least two barcodes of the plurality of barcodes may comprise different molecular beacon sequences. The barcode may comprise a cell beacon, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0042] In some embodiments, the plurality of barcodes are immobilized on particles. At least one barcode of the plurality of barcodes can be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof. The particles can be degradable. The particles can be beads. The beads can be selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise at least 10,000 barcodes. In some embodiments, the barcodes of the particles may comprise molecular beacon sequences selected from at least 1,000 or 10,000 different molecular beacon sequences. The molecular beacon sequences of the barcodes may comprise random sequences.

[0043] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes may include contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0044] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data may include sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

[0045] In some embodiments, barcoding sample-indexing oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides comprises probabilistic barcoding sample-indexing oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded sample-indexing oligonucleotides. Identifying the sample origin of the at least one cell may comprise identifying the presence or absence of a sample-indexing sequence of at least one sample-indexing oligonucleotide of the plurality of sample-indexing compositions. Identifying the presence or absence of a sample-indexing sequence may comprise replicating at least one sample-indexing oligonucleotide to generate a plurality of replicated sample-indexing oligonucleotides; obtaining sequencing data of the plurality of replicated sample-indexing oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of the replicated sample-indexing oligonucleotide of the plurality of sample-indexing oligonucleotides that corresponds to the at least one barcoded sample-indexing oligonucleotide in the sequencing data.

[0046] In some embodiments, replicating at least one sample-indexed oligonucleotide to generate a plurality of replicated sample-indexed oligonucleotides comprises ligating a replication adapter to the at least one barcoded sample-indexed oligonucleotide before replicating the at least one barcoded sample-indexed oligonucleotide. Replicating at least one barcoded sample-indexed oligonucleotide may comprise replicating the at least one barcoded sample-indexed oligonucleotide using a replication adapter ligated to the at least one barcoded sample-indexed oligonucleotide to generate a plurality of replicated sample-indexed oligonucleotides.

[0047] In some embodiments, replicating at least one sample-indexing oligonucleotide to generate a plurality of replicated sample-indexing oligonucleotides comprises: contacting a capture probe with the at least one sample-indexing oligonucleotide to generate a capture probe hybridized to the sample-indexing oligonucleotide prior to replicating the at least one barcoded sample-indexing oligonucleotide; and extending the capture probe hybridized to the sample-indexing oligonucleotide to generate a sample-indexing oligonucleotide associated with the capture probe. Replicating at least one sample-indexing oligonucleotide may comprise replicating the sample-indexing oligonucleotide associated with the capture probe to generate a plurality of replicated sample-indexing oligonucleotides.

[0048] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, each of the plurality of barcodes comprising a cell label, and at least two barcodes of the plurality of barcodes comprising an identical cell label sequence; and acquiring sequencing data for the barcoded targets. Identifying the sample origin of at least one barcoded sample-indexing oligonucleotide may include identifying the sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded sample-indexing oligonucleotide. Barcoding the plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target-binding regions of the barcodes; and reverse-transcribe the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. The method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to acquiring sequencing data for the plurality of barcoded targets. Amplifying the barcoded target to generate a plurality of amplified barcoded targets may include amplifying the barcoded target by polymerase chain reaction (PCR). Barcoding the plurality of cellular targets with the plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding the plurality of cellular targets with the plurality of stochastic barcodes to generate a plurality of stochastically barcoded targets.

[0049] In some embodiments, each of the plurality of sample indexing compositions comprises an antigen-binding reagent. The sample indexing sequences of the sample indexing oligonucleotides associated with two or more antigen-binding reagents may be identical. The sample indexing sequences of the sample indexing oligonucleotides associated with two or more antigen-binding reagents may comprise different sequences. Each of the plurality of sample indexing compositions may comprise two or more antigen-binding reagents.

[0050] Disclosed herein are a plurality of sample indexing compositions. Each of the plurality of sample indexing compositions may comprise two or more antigen binding reagents. Each of the two or more antigen binding reagents may be associated with a sample indexing oligonucleotide. At least one of the two or more antigen binding reagents may be capable of specifically binding to at least one antigen target. The sample indexing oligonucleotide may comprise a sample indexing sequence for identifying the sample origin of one or more cells of the sample. The sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences.

[0051] In some embodiments, the sample indexing sequences comprise a nucleotide sequence that is 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, or at least 128 nucleotides in length. The sample indexing sequences of at least 10, 100, or 1000 sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0052] In some embodiments, the antigen-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample indexing oligonucleotide may be attached to the antigen-binding reagent via a linker. At least one sample indexing oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the antigen-binding reagent molecule. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0053] In some embodiments, the sample indexing oligonucleotide is not homologous to a genomic sequence of a species. The sample indexing oligonucleotide may comprise a molecular beacon sequence, a poly(A) tract, or a combination thereof. In some embodiments, at least one sample of the plurality of samples may comprise a single cell, multiple cells, a tissue, a tumor sample, or any combination thereof. The sample may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0054] In some embodiments, the antigen target is or includes a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, or any combination thereof. The antigen target may be selected from a group comprising 10 to 100 different antigen targets. An antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having the same sequence. An antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences. A sample-indexing composition of the plurality of sample-indexing compositions may include a second antigen-binding reagent that is not bound to a sample-indexing oligonucleotide. The antigen-binding reagent and the second antigen-binding reagent may be identical.

[0055] Disclosed herein is a control particle composition. In some embodiments, the control particle composition comprises a plurality of control particle oligonucleotides associated with control particles, each of the plurality of control particle oligonucleotides comprising a control barcode sequence and a poly(dA) region. At least two of the plurality of control particle oligonucleotides may comprise different control barcode sequences. The control particle oligonucleotide may comprise a molecular beacon sequence. The control particle oligonucleotide may comprise a binding site for a universal primer.

[0056] In some embodiments, the control barcode sequence is at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The control particle oligonucleotide can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. At least 5, 10, 100, 1000, or more control barcode sequences of a plurality of control particle oligonucleotides can be identical. At least 10, 100, 1000, or more control barcode sequences of a plurality of control particle oligonucleotides can be identical. At least 3, 5, 10, 100, or more of the plurality of control particle oligonucleotides can contain different control barcode sequences.

[0057] In some embodiments, the plurality of control particle oligonucleotides comprises a plurality of first control particle oligonucleotides each comprising a first control barcode sequence and a plurality of second control particle oligonucleotides each comprising a second control barcode sequence, wherein the first control barcode sequence and the second control barcode sequence have different sequences. The number of the plurality of first control particle oligonucleotides and the number of the plurality of second control particle oligonucleotides can be approximately the same. The number of the plurality of first control particle oligonucleotides and the number of the plurality of second control particle oligonucleotides can be different. The number of the plurality of first control particle oligonucleotides can be at least 2-fold, 10-fold, 100-fold, or more greater than the number of the plurality of second control particle oligonucleotides.

[0058] In some embodiments, the control barcode sequence is not homologous to the genome sequence of a species. The control barcode sequence can be homologous to the genome sequence of a species. The species can be a non-mammalian species. The non-mammalian species can be a phage species. The phage species can be T7 phage, PhiX phage, or a combination thereof.

[0059] In some embodiments, at least one of the plurality of control particle oligonucleotides is associated with the control particle via a linker. At least one of the plurality of control particle oligonucleotides may include a linker. The linker may include a chemical group. The chemical group may be reversibly bound to at least one of the plurality of control particle oligonucleotides. The chemical group may include a UV light-cleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0060] In some embodiments, the diameter of the control particle is about 1-1000 micrometers, about 10-100 micrometers, 7.5 micrometers, or a combination thereof.

[0061] In some embodiments, the plurality of control particle oligonucleotides are immobilized on the control particle. The plurality of control particle oligonucleotides may be partially immobilized on the control particle. The plurality of control particle oligonucleotides may be encapsulated in the control particle. The plurality of control particle oligonucleotides may be partially encapsulated in the control particle. The control particle may be disintegrable. The control particle may be a bead. The bead may include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The control particles may comprise polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof. The control particles may comprise disintegrating hydrogel particles.

[0062] In some embodiments, the control particle is associated with a detectable moiety. The control particle oligonucleotide can be associated with a detectable moiety.

[0063] In some embodiments, the control particle is associated with a plurality of first protein-binding reagents, at least one of which is associated with one of a plurality of control particle oligonucleotides. The first protein-binding reagent may include a first antibody. The control particle oligonucleotide may be bound to the first protein-binding reagent via a linker. The first control particle oligonucleotide may include a linker. The linker may include a chemical group. The chemical group may be reversibly bound to the first protein-binding reagent. The chemical group may include a UV light-cleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0064] In some embodiments, the first protein binding reagent is associated with two or more of the plurality of control particle oligonucleotides having the same control barcode sequence. The first protein binding reagent can be associated with two or more of the plurality of control particle oligonucleotides having different control barcode sequences. In some embodiments, at least one of the plurality of first protein binding reagents is not associated with any of the plurality of control particle oligonucleotides. The first protein binding reagent associated with the control particle oligonucleotide and the first protein binding reagent not associated with any control particle oligonucleotide can be the same protein binding reagent.

[0065] In some embodiments, the control particle is associated with a plurality of second protein-binding reagents. At least one of the plurality of second protein-binding reagents can be associated with one of a plurality of control particle oligonucleotides. The control particle oligonucleotide associated with the first protein-binding reagent and the control particle oligonucleotide associated with the second protein-binding reagent can include different control barcode sequences. The first protein-binding reagent and the second protein-binding reagent can be the same protein-binding reagent.

[0066] In some embodiments, the first protein binding reagent is associated with a partner binding reagent, and the first protein binding reagent is associated with the control particle using the partner binding reagent. The partner binding reagent may include a partner antibody. The partner antibody may include an anti-cat antibody, an anti-chicken antibody, an anti-cow antibody, an anti-dog antibody, an anti-donkey antibody, an anti-goat antibody, an anti-guinea pig antibody, an anti-hamster antibody, an anti-horse antibody, an anti-human antibody, an anti-llama antibody, an anti-monkey antibody, an anti-mouse antibody, an anti-pig antibody, an anti-rabbit antibody, an anti-rat antibody, an anti-sheep antibody, or a combination thereof. The partner antibody may include immunoglobulin G (IgG), an F(ab') fragment, an F(ab')2 fragment, a combination thereof, or a fragment thereof.

[0067] In some embodiments, the first protein-binding reagent can be associated with a detectable moiety. The second protein-binding reagent can be associated with a detectable moiety.

[0068] The present disclosure includes a method for determining the number of targets. In some embodiments, the method includes using a plurality of probabilistic barcodes to probabilistically barcode a plurality of targets and a plurality of control particle oligonucleotides of a cell among a plurality of cells to generate a plurality of probabilistically barcoded targets and a plurality of probabilistically barcoded control particle oligonucleotides, wherein each of the plurality of probabilistic barcodes includes a cell label sequence, a molecular label sequence, and a target binding region, wherein the molecular label sequences of at least two of the plurality of probabilistic barcodes include different sequences, and at least two of the plurality of probabilistic barcodes include the same cell label sequence, and the control particle composition includes control particles associated with a plurality of control particle oligonucleotides, wherein each of the plurality of control particle oligonucleotides includes a pseudo target region including a control barcode sequence and a sequence substantially complementary to the target binding region of at least one of the plurality of probabilistic barcodes. The method may include obtaining sequencing data for the plurality of probabilistically barcoded targets and the plurality of probabilistically barcoded control particle oligonucleotides; and counting the number of molecular label sequences having distinguishable sequences associated with the plurality of control particle oligonucleotides having the control barcode sequence in the sequencing data. The method may include, for at least one target of the plurality of targets: counting the number of molecular beacon sequences having distinguishable sequences associated with the target in the sequencing data; and estimating the number of targets, wherein the estimated number of targets correlates with the number of molecular beacon sequences having distinguishable sequences associated with the counted target and the number of molecular beacon sequences having distinguishable sequences associated with a control barcode sequence.

[0069] In some embodiments, the pseudo target region comprises a poly(dA) region. The pseudo target region may comprise a subsequence of the target. In some embodiments, the control barcode sequence may be at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The control particle oligonucleotide may be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or any combination thereof. At least 5, 10, 100, 1000, or more control barcode sequences of the plurality of control particle oligonucleotides may be identical. At least 3, 5, 10, 100, or more of the plurality of control particle oligonucleotides may comprise different control barcode sequences.

[0070] In some embodiments, the plurality of control particle oligonucleotides includes a plurality of first control particle oligonucleotides each including a first control barcode sequence and a plurality of second control particle oligonucleotides each including a second control barcode sequence. The first control barcode sequence and the second control barcode sequence may have different sequences. The number of the plurality of first control particle oligonucleotides and the number of the plurality of second control particle oligonucleotides may be approximately the same. The number of the plurality of first control particle oligonucleotides and the number of the plurality of second control particle oligonucleotides may be different. The number of the plurality of first control particle oligonucleotides may be at least 2-fold, 10-fold, 100-fold, or more than the number of the plurality of second control particle oligonucleotides.

[0071] In some embodiments, counting the number of molecular label sequences having distinguishable sequences associated with a plurality of control particle oligonucleotides having control barcode sequences in the sequencing data comprises: counting the number of molecular label sequences having distinguishable sequences associated with a first control barcode sequence in the sequencing data; and counting the number of molecular label sequences having distinguishable sequences associated with the second control barcode sequence in the sequencing data. The number of estimated targets can be correlated with the number of molecular label sequences having distinguishable sequences associated with the counted targets, the number of molecular label sequences having distinguishable sequence sequences associated with the first control barcode sequence, and the number of molecular label sequences having distinguishable sequences associated with the second control barcode sequence. The number of estimated targets can be correlated with the number of molecular label sequences having distinguishable sequences associated with the counted targets, the number of molecular label sequences having distinguishable sequences associated with the control barcode sequence, and the number of a plurality of control particle oligonucleotides comprising control barcode sequences. The estimated number of targets can be correlated with the number of molecular beacon sequences with identifiable sequences associated with the counted targets, and the ratio of the number of multiple control particle oligonucleotides containing control barcode sequences to the number of molecular beacon sequences with identifiable sequences associated with the control barcode sequences.

[0072] In some embodiments, the control particle oligonucleotide is not homologous to the genomic sequence of the cell. The control particle oligonucleotide may not be homologous to the genomic sequence of the species. The control particle oligonucleotide may be homologous to the genomic sequence of one species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species may be T7 phage, PhiX phage, or a combination thereof.

[0073] In some embodiments, the control particle oligonucleotide can be bound to the control particle via a linker. At least one of the plurality of control particle oligonucleotides can be associated with the control particle via a linker. At least one of the plurality of control particle oligonucleotides can include a linker. A chemical group can be reversibly bound to at least one of the plurality of control particle oligonucleotides. The chemical group can include a UV light-cleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0074] In some embodiments, the diameter of the control particle is about 1-1000 micrometers, about 10-100 micrometers, about 7.5 micrometers, or a combination thereof. A plurality of control particle oligonucleotides are immobilized on the control particle. A plurality of control particle oligonucleotides may be partially immobilized on the control particle. A plurality of control particle oligonucleotides may be encapsulated in the control particle. A plurality of control particle oligonucleotides may be partially encapsulated in the control particle.

[0075] In some embodiments, the method includes releasing at least one of the plurality of control particle oligonucleotides from the control particle prior to stochastically barcoding the plurality of target and control particles and the plurality of control particle oligonucleotides.

[0076] In some embodiments, the control particles are disintegrating. The control particles may be control particle beads. The control particle beads may include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The control particles may include polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof. The control particles may include disintegrating hydrogel particles.

[0077] In some embodiments, the control particle is associated with a detectable moiety. The control particle oligonucleotide can be associated with a detectable moiety.

[0078] In some embodiments, the control particle is associated with a plurality of first protein-binding reagents, at least one of which may be associated with one of a plurality of control particle oligonucleotides. The first protein-binding reagent may comprise a first antibody. The control particle oligonucleotide may be bound to the first protein-binding reagent via a linker. The first control particle oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly bound to the first protein-binding reagent. The chemical group may comprise a UV light-cleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0079] In some embodiments, a first protein binding reagent can be associated with two or more of a plurality of control particle oligonucleotides having the same control barcode sequence. A first protein binding reagent can be associated with two or more of a plurality of control particle oligonucleotides having different control barcode sequences. At least one of the plurality of first protein binding reagents cannot be associated with any of the plurality of control particle oligonucleotides. A first protein binding reagent associated with a control particle oligonucleotide and a first protein binding reagent not associated with any control particle oligonucleotide can be the same protein binding reagent. A control particle can be associated with a plurality of second protein binding reagents. At least one of the plurality of second protein binding reagents can be associated with one of the plurality of control particle oligonucleotides. The control particle oligonucleotide associated with the first protein binding reagent and the control particle oligonucleotide associated with the second protein binding reagent can include different control barcode sequences. The first protein binding reagent and the second protein binding reagent can be the same protein binding reagent.

[0080] In some embodiments, the first protein binding reagent is associated with a partner binding reagent, and the first protein binding reagent is associated with the control particle using the partner binding reagent. The partner binding reagent may include a partner antibody. The partner antibody may include an anti-cat antibody, an anti-chicken antibody, an anti-cow antibody, an anti-dog antibody, an anti-donkey antibody, an anti-goat antibody, an anti-guinea pig antibody, an anti-hamster antibody, an anti-horse antibody, an anti-human antibody, an anti-llama antibody, an anti-monkey antibody, an anti-mouse antibody, an anti-pig antibody, an anti-rabbit antibody, an anti-rat antibody, an anti-sheep antibody, or a combination thereof. The partner antibody may include immunoglobulin G (IgG), an F(ab') fragment, an F(ab')2 fragment, a combination thereof, or a fragment thereof.

[0081] In some embodiments, the first protein-binding reagent can be associated with a detectable moiety. The second protein-binding reagent can be associated with a detectable moiety.

[0082] In some embodiments, the probabilistic barcode comprises a binding site for a universal primer. The target binding region may comprise a poly(dT) region.

[0083] In some embodiments, the plurality of probabilistic barcodes are associated with a bar-coding particle. For example, at least one of the plurality of probabilistic barcodes may be immobilized on a bar-coding particle. At least one of the plurality of probabilistic barcodes may be partially immobilized on a bar-coding particle. At least one of the plurality of probabilistic barcodes may be encapsulated in a bar-coding particle. At least one of the plurality of probabilistic barcodes may be partially encapsulated in a bar-coding particle.

[0084] In some embodiments, the bar-coding particles are disintegrating. The bar-coding particles may be bar-coding beads. The bar-coding beads may include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The bar-coding particles may include polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof. The bar-coding particles may include disintegrating hydrogel particles.

[0085] In some embodiments, the stochastic barcode of the barcoding particle comprises a molecular label sequence selected from at least 1,000, 10,000, or more different molecular label sequences. In some embodiments, the molecular label sequence of the stochastic barcode comprises a random sequence. In some embodiments, the barcoding particle comprises at least 10,000 stochastic barcodes.

[0086] In some embodiments, the step of stochastically barcoding the plurality of target and plurality of control particle oligonucleotides using the plurality of stochastic barcodes comprises contacting the plurality of stochastic barcodes with targets of the plurality of targets and control particle oligonucleotides of the plurality of control particle oligonucleotides to generate stochastic barcodes hybridized to the target and control particle oligonucleotides; and extending the stochastic barcodes hybridized to the target and control particle oligonucleotides to generate the plurality of stochastically barcoded targets and the plurality of stochastically barcoded control particle oligonucleotides. Extending the stochastic barcodes may comprise extending the stochastic barcodes using a DNA polymerase, a reverse transcriptase, or a combination thereof.

[0087] In some embodiments, the method includes amplifying a plurality of stochastically barcoded targets and a plurality of stochastically barcoded control particle oligonucleotides to generate a plurality of amplicons. Amplifying the plurality of stochastically barcoded targets and a plurality of stochastically barcoded control particle oligonucleotides may include amplifying at least a portion of the molecular beacon sequences and at least a portion of the control particle oligonucleotides, or at least a portion of the molecular beacon sequences and at least a portion of the control particle oligonucleotides, using polymerase chain reaction (PCR). Obtaining sequencing data may include obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data may include sequencing at least a portion of the molecular beacon sequences and at least a portion of the control particle oligonucleotides, or at least a portion of the molecular beacon sequences and at least a portion of the control particle oligonucleotides.

[0088] Also disclosed herein are kits for sequence control. In some embodiments, the kits include a control particle composition comprising a plurality of control particle oligonucleotides associated with control particles, each of the plurality of control particle oligonucleotides comprising a control barcode sequence and a poly(dA) region.

[0089] In some embodiments, at least two of the plurality of control particle oligonucleotides comprise different control barcode sequences. In some embodiments, the control barcode sequences can be at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The control particle oligonucleotides can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or any combination thereof. At least 5, 10, 100, 1000, or more of the control particle oligonucleotides in the plurality of control particle oligonucleotides can be identical. At least 3, 5, 10, 100, or more of the control particle oligonucleotides in the plurality of control particle oligonucleotides can comprise different control barcode sequences.

[0090] In some embodiments, the plurality of control particle oligonucleotides includes a plurality of first control particle oligonucleotides each including a first control barcode sequence and a plurality of second control particle oligonucleotides each including a second control barcode sequence. The first control barcode sequence and the second control barcode sequence may have different sequences. The number of the plurality of first control particle oligonucleotides and the number of the plurality of second control particle oligonucleotides may be approximately the same. The number of the plurality of first control particle oligonucleotides and the number of the plurality of second control particle oligonucleotides may be different. The number of the plurality of first control particle oligonucleotides may be at least 2-fold, 10-fold, 100-fold, or more than the number of the plurality of second control particle oligonucleotides.

[0091] In some embodiments, the control particle oligonucleotide is not homologous to the genomic sequence of the cell. The control particle oligonucleotide may not be homologous to the genomic sequence of the species. The control particle oligonucleotide may be homologous to the genomic sequence of one species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species may be T7 phage, PhiX phage, or a combination thereof.

[0092] In some embodiments, the control particle oligonucleotide can be bound to the control particle via a linker. At least one of the plurality of control particle oligonucleotides can be associated with the control particle via a linker. At least one of the plurality of control particle oligonucleotides can include a linker. A chemical group can be reversibly bound to at least one of the plurality of control particle oligonucleotides. The chemical group can include a UV light-cleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0093] In some embodiments, the diameter of the control particle is about 1-1000 micrometers, about 10-100 micrometers, about 7.5 micrometers, or a combination thereof. A plurality of control particle oligonucleotides are immobilized on the control particle. A plurality of control particle oligonucleotides may be partially immobilized on the control particle. A plurality of control particle oligonucleotides may be encapsulated in the control particle. A plurality of control particle oligonucleotides may be partially encapsulated in the control particle.

[0094] In some embodiments, the kit includes a plurality of barcodes. A barcode of the plurality of barcodes can include a target binding region and a molecular beacon sequence, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes can include different molecular beacon sequences. The barcodes can include a cell beacon sequence, a binding site for a universal primer, or any combination thereof. The target binding region includes a poly(dT) region.

[0095] In some embodiments, a plurality of barcodes can be associated with a bar-coding particle. At least one barcode of the plurality of barcodes can be immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes can be partially immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes can be encapsulated in the bar-coding particle. At least one barcode of the plurality of barcodes can be partially encapsulated in the bar-coding particle. The bar-coding particle can be disintegrating. The bar-coding particle can be a second bead. The bead can be or include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The bar-coding particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The bar-coding particles may comprise degradable hydrogel particles.

[0096] In some embodiments, the barcode of the bar-coding particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. The molecular beacon sequence of the barcode may comprise a random sequence. The bar-coding particle may comprise at least 10,000 barcodes. The kit may comprise a DNA polymerase. The kit may comprise reagents for polymerase chain reaction (PCR).

[0097] The method disclosed herein for cell identification includes contacting a first plurality of cells and a second plurality of cells with two sample indexing compositions, respectively, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more protein targets, each of the two sample indexing compositions comprising a protein binding reagent associated with a sample indexing oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the one or more protein targets, the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprising different sequences; and identifying the sample indexing sequences using a plurality of barcodes. The method may include barcoding oligonucleotides to generate a plurality of barcoded sample-indexing oligonucleotides, each of the plurality of barcodes comprising a cell label sequence, a molecular label sequence, and a target-binding region, wherein the molecular label sequences of at least two of the plurality of barcodes comprise different sequences, and at least two of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data for the plurality of barcoded sample-indexing oligonucleotides; identifying cell label sequences associated with two or more sample-indexing sequences in the acquired sequencing data; and removing sequencing data associated with the cell label sequences from the acquired sequencing data. In some embodiments, the sample-indexing oligonucleotides comprise a molecular label sequence, a binding site for a universal primer, or a combination thereof. As described herein, the first plurality of cells can be obtained from or derived from a different tissue or organ than the second plurality of cells, and the first plurality of cells and the second plurality of cells can be derived from the same or different subjects (e.g., mammals). For example, the first plurality of cells can be obtained or derived from a different human subject than the second plurality of cells, hi some embodiments, the first plurality of cells and the second plurality of cells are obtained or derived from different tissues of the same human subject.

[0098] In some embodiments, contacting the first plurality of cells and the second plurality of cells with two sample indexing compositions, respectively, comprises: contacting the first plurality of cells with a first sample indexing composition of the two sample indexing compositions; and contacting the first plurality of cells with a second sample indexing composition of the two sample indexing compositions.

[0099] As described herein, a sample indexing sequence can be, for example, at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. A sample indexing oligonucleotide can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. In some embodiments, the sample indexing sequences of at least 10, 100, 1000, or more sample indexing compositions of a plurality of sample indexing compositions comprise different sequences.

[0100] In some embodiments, the protein binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample indexing oligonucleotide may be attached to the protein binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly attached to the protein binding reagent. The chemical group may comprise a UV photocleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0101] In some embodiments, at least one sample of the plurality of samples comprises a single cell, and at least one of the one or more protein targets may be on the cell surface.

[0102] In some embodiments, the method includes removing unbound sample indexing compositions of the two sample indexing compositions. Removing unbound sample indexing compositions may include washing the cells of the first plurality of cells and the second plurality of cells with a wash buffer. Removing unbound sample indexing compositions may include using flow cytometry to select cells bound to a protein-binding reagent of at least one of the two sample indexing compositions. In some embodiments, the method includes lysing one or more cells from each of the plurality of samples.

[0103] In some embodiments, the sample indexing oligonucleotide is configured to be cleavable or non-cleavable from the protein binding reagent. The method may include cleaving the sample indexing oligonucleotide from the protein binding reagent. Cleavage of the sample indexing oligonucleotide may include cleaving the sample indexing oligonucleotide from the protein binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof.

[0104] In some embodiments, the sample-indexing oligonucleotide is not homologous to any genomic sequence of one or more cells. The control barcode sequence may not be homologous to the genomic sequence of a species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species may be T7 phage, PhiX phage, or a combination thereof.

[0105] In some embodiments, one sample of the plurality of samples comprises a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes. The barcode may comprise a target binding region comprising the capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(dA) region.

[0106] In some embodiments, the protein target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The protein target may be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The protein target may be or include a lipid, a carbohydrate, or any combination thereof. The protein target may be selected from a group comprising 10 to 100 different protein targets.

[0107] In some embodiments, a protein binding reagent is associated with two or more sample indexing oligonucleotides having the same sequence. A protein binding reagent may be associated with two or more sample indexing oligonucleotides having different sample indexing sequences. A sample indexing composition of the plurality of sample indexing compositions may include a second protein binding reagent that is not associated with a sample indexing oligonucleotide. The protein binding reagent and the second protein binding reagent may be the same.

[0108] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences. The barcode may comprise a cell beacon sequence, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0109] In some embodiments, a plurality of barcodes can be associated with a particle. At least one barcode of the plurality of barcodes can be immobilized on a particle. At least one barcode of the plurality of barcodes can be partially immobilized on a particle. At least one barcode of the plurality of barcodes can be encapsulated in a particle. At least one barcode of the plurality of barcodes can be partially encapsulated in a particle. The particle can be disintegrable. The particle can be a bead. The bead can be or include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise collapsible hydrogel particles.

[0110] In some embodiments, the protein binding reagent is associated with a detectable moiety. In some embodiments, the particle is associated with a detectable moiety. The sample-indexing oligonucleotide is associated with an optical moiety.

[0111] In some embodiments, the barcode of a particle can comprise a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. The molecular beacon sequence of the barcode can comprise a random sequence. The particle can comprise at least 10,000 barcodes.

[0112] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0113] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of a molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). In some embodiments, acquiring sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include acquiring sequencing data for the plurality of amplicons. Acquiring the sequencing data includes sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides. In some embodiments, identifying the sample origin of the at least one cell includes identifying the sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded, sample-indexing oligonucleotide.

[0114] In some embodiments, barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

[0115] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; and obtaining sequencing data for the barcoded targets. Barcoding the plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets can include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets.

[0116] In some embodiments, the method includes amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding the plurality of cellular targets with the plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding the plurality of cellular targets with the plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0117] Also disclosed herein are methods and compositions that can be used for sequencing control. In some embodiments, a method for sequencing control includes contacting one or more cells of a plurality of cells with a control composition of a plurality of control compositions, wherein the one cell of the plurality of cells comprises a plurality of targets and a plurality of protein targets, each of the plurality of control compositions comprising a protein binding reagent associated with a control oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the plurality of protein targets, and the control oligonucleotide comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to the target binding region of at least one of the plurality of barcodes; barcoding the control oligonucleotide using the plurality of barcodes to generate a plurality of barcoded control oligonucleotides, wherein each of the plurality of barcodes comprises a cell label sequence, a molecular label sequence, and a target binding region, wherein the molecular label sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded control oligonucleotides; and determining at least one characteristic of the one or more cells using at least one characteristic of the plurality of barcoded control oligonucleotides in the sequencing data. In some embodiments, the pseudo-target region comprises a poly(dA) region.

[0118] In some embodiments, the control barcode sequence is at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The control particle oligonucleotide can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. At least 2, 10, 100, 1000, or more of the control barcode sequences of the plurality of control particle oligonucleotides can be identical. At least 2, 10, 100, 1000, or more of the plurality of control particle oligonucleotides can contain different control barcode sequences.

[0119] In some embodiments, determining at least one characteristic of the one or more cells includes: determining the number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides in the sequencing data; and determining the number of one or more cells using the number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides. The method may include determining a single-cell capture efficiency based on the determined number of the one or more cells. The method may include determining a single-cell capture efficiency based on a ratio of the determined number of the one or more cells to the number of the plurality of cells.

[0120] In some embodiments, determining at least one characteristic of one or more cells using features of a plurality of barcoded control oligonucleotides in the sequencing data may include: determining, for each cell marker in the sequencing data, the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence; and determining the number of one or more cells using the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence. Determining the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence may include, for each cell marker in the sequencing data, determining the number of molecular marker sequences having the most distinguishable sequences associated with the cell marker and the control barcode sequence. Determining the number of one or more cells using the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence may include creating a plot of the number of cell markers and the number of molecular marker sequences having the most distinguishable sequences in the sequencing data associated with the number of molecular marker sequences having the most distinguishable sequences; and determining a cutoff in the plot as the number of one or more cells.

[0121] In some embodiments, the control oligonucleotide is not homologous to any genomic sequence of the plurality of cells. The control oligonucleotide can be homologous to a genomic sequence of one species. The species can be a non-mammalian species. The non-mammalian species can be a phage species. The phage species can be T7 phage, PhiX phage, or a combination thereof.

[0122] In some embodiments, the method includes releasing the control oligonucleotide from the protein-binding reagent before barcoding the control oligonucleotide. In some embodiments, the method includes removing unbound control compositions of the plurality of control compositions. Removing unbound control compositions may include washing one or more cells of the plurality of cells with a wash buffer. Removing unbound sample-indexing compositions may include using flow cytometry to select cells bound to at least one protein-binding reagent of the control composition.

[0123] In some embodiments, at least one of the multiple protein targets is on a cell surface. At least one of the multiple protein targets may include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The protein binding reagent may include an antibody. The control oligonucleotide may be attached to the protein binding reagent via a linker. The control oligonucleotide may include a linker. The linker may include a chemical group. The chemical group may be reversibly attached to the first protein binding reagent. The chemical group may include a UV photocleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0124] In some embodiments, the protein binding reagent is associated with two or more control oligonucleotides having the same control barcode sequence. The protein binding reagent may be associated with two or more control oligonucleotides having different identical control barcode sequences. In some embodiments, the second protein binding reagent of the plurality of control compositions is not associated with a control oligonucleotide. The protein binding reagent and the second protein binding reagent may be the same.

[0125] In some embodiments, the barcode comprises a binding site for a universal primer. The target binding region may comprise a poly(dT) region. In some embodiments, the plurality of barcodes is associated with a bar-coding particle. At least one barcode of the plurality of barcodes may be immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes may be partially immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes is encapsulated in the bar-coding particle. At least one barcode of the plurality of barcodes is partially encapsulated in the bar-coding particle. The bar-coding particle may be disintegrable. The bar-coding particle may be a bar-coding bead. The bar-coding beads may include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The bar-coding particles may include polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof. The bar-coding particles may include degradable hydrogel particles.

[0126] In some embodiments, the bar-coded particle is associated with an optical moiety. The control oligonucleotide can be associated with an optical moiety.

[0127] In some embodiments, the barcode of the barcode particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. In some embodiments, the molecular beacon sequence of the barcode comprises a random sequence. In some embodiments, the barcode particle comprises at least 10,000 barcodes.

[0128] In some embodiments, barcoding the control oligonucleotides comprises barcoding the control oligonucleotides with a plurality of stochastic barcodes to generate a plurality of stochastically barcoded control oligonucleotides. In some embodiments, barcoding the plurality of control oligonucleotides with a plurality of barcodes comprises contacting the plurality of barcodes with a plurality of control oligonucleotides of a control composition to generate barcodes hybridized to the control oligonucleotides; and extending the stochastic barcodes hybridized to the control oligonucleotides to generate a plurality of barcoded control oligonucleotides. Extending the barcodes may comprise extending the barcodes using a DNA polymerase, a reverse transcriptase, or a combination thereof. In some embodiments, the method comprises amplifying the plurality of barcoded control oligonucleotides to generate a plurality of amplicons. Amplifying the plurality of barcoded control oligonucleotides may comprise amplifying at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotides using polymerase chain reaction (PCR). In some embodiments, obtaining sequencing data comprises obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data can include sequencing at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotide.

[0129] Disclosed herein are methods for sequencing control. In some embodiments, the method includes contacting one or more cells of a plurality of cells with a control composition from a plurality of control compositions, wherein the one cell of the plurality of cells comprises a plurality of targets and a plurality of binding targets, each of the plurality of control compositions comprising a cellular component binding reagent associated with a control oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the plurality of binding targets, and the control oligonucleotide comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to the target binding region of at least one of the plurality of barcodes; barcoding the control oligonucleotide using the plurality of barcodes to generate a plurality of barcoded control oligonucleotides, each of the plurality of barcodes comprising a cell label sequence, a molecular label sequence, and a target binding region, wherein the molecular label sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded control oligonucleotides; and determining at least one characteristic of the one or more cells using at least one characteristic of the plurality of barcoded control oligonucleotides in the sequencing data. In some embodiments, the pseudo-target region comprises a poly(dA) region.

[0130] In some embodiments, the control barcode sequence is at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The control particle oligonucleotide can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. At least 2, 10, 100, 1000, or more of the control barcode sequences of the plurality of control particle oligonucleotides can be identical. At least 2, 10, 100, 1000, or more of the plurality of control particle oligonucleotides can contain different control barcode sequences.

[0131] In some embodiments, determining at least one characteristic of the one or more cells comprises: determining a number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides in the sequencing data; and determining a number of the one or more cells using the number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides. In some embodiments, the method comprises determining a single-cell capture efficiency based on the determined number of the one or more cells. In some embodiments, the method comprises determining a single-cell capture efficiency based on a ratio of the determined number of the one or more cells and the number of the plurality of cells.

[0132] In some embodiments, determining at least one characteristic of the one or more cells includes, for each cell marker in the sequencing data, determining the number of molecular marker sequences and control barcode sequences having distinguishable sequences associated with the cell marker; and determining the number of one or more cells using the number of molecular marker sequences and control barcode sequences having distinguishable sequences associated with the cell marker. Determining the number of molecular marker sequences and control barcode sequences having distinguishable sequences associated with the cell marker may include, for each cell marker in the sequencing data, determining the number of molecular marker sequences and control barcode sequences having distinguishable sequences associated with the cell marker with the highest number of distinguishable sequences. Determining the number of one or more cells using the number of molecular marker sequences and control barcode sequences having distinguishable sequences associated with the cell marker may include creating a plot of the number of molecular marker sequences having the highest number of distinguishable sequences using the number of cell markers in the sequencing data associated with the number of molecular marker sequences having the highest number of distinguishable sequences; and determining a cutoff in the plot as the number of one or more cells.

[0133] In some embodiments, the control oligonucleotide is not homologous to any genomic sequence of the plurality of cells. The control oligonucleotide can be homologous to a genomic sequence of one species. The species can be a non-mammalian species. The non-mammalian species can be a phage species. The phage species can be T7 phage, PhiX phage, or a combination thereof.

[0134] In some embodiments, the method includes releasing the control oligonucleotide from the cellular component-binding reagent before barcoding the control oligonucleotide. At least one of the multiple binding targets can be expressed on a cell surface. At least one of the multiple binding targets can include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The cellular component-binding reagent can include a cell surface-binding reagent, an antibody, a tetramer, an aptamer, a protein scaffold, an invasion, or a combination thereof.

[0135] In some embodiments, the binding target of the cellular component binding reagent is selected from a group containing 10 to 100 different binding targets. The binding target of the cellular component binding reagent may include carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, integrins, intracellular proteins, or any combination thereof. The control oligonucleotide may be attached to the cellular component binding reagent via a linker. The control oligonucleotide may include a linker. The linker may include a chemical group. The chemical group may be reversibly attached to the first cellular component binding reagent. The chemical group may include a UV light-cleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0136] In some embodiments, a cellular component binding reagent can be associated with two or more control oligonucleotides having the same control barcode sequence. A cellular component binding reagent can be associated with two or more control oligonucleotides having different identical control barcode sequences. In some embodiments, a second cellular component binding reagent of the plurality of control compositions is not associated with a control oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent can be the same.

[0137] In some embodiments, the barcode comprises a binding site for a universal primer, hi some embodiments, the target binding region comprises a poly(dT) region.

[0138] In some embodiments, the plurality of barcodes are associated with a bar-coding particle. At least one barcode of the plurality of barcodes may be immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes may be partially immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes may be encapsulated in the bar-coding particle. At least one barcode of the plurality of barcodes may be partially encapsulated in the bar-coding particle. The bar-coding particle may be disintegrable. The bar-coding particle may be a bar-coding bead. In some embodiments, the bar-coding bead comprises sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The bar-coding particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and combinations thereof. The bar-coding particles may comprise collapsible hydrogel particles. The bar-coding particles may be associated with an optical moiety.

[0139] In some embodiments, the control oligonucleotide can be associated with an optical moiety. In some embodiments, the barcode of the barcode particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. In some embodiments, the molecular beacon sequence of the barcode comprises a random sequence. The barcode particle can comprise at least 10,000 barcodes.

[0140] In some embodiments, barcoding the control oligonucleotides comprises barcoding the control oligonucleotides with a plurality of stochastic barcodes to generate a plurality of stochastically barcoded control oligonucleotides. Barcoding the control oligonucleotides with a plurality of barcodes may comprise contacting the plurality of barcodes with a plurality of control oligonucleotides of a control composition to generate barcodes hybridized to the control oligonucleotides; and extending the stochastic barcodes hybridized to the control oligonucleotides to generate a plurality of barcoded control oligonucleotides. Extending the barcodes may comprise extending the barcodes using a DNA polymerase, a reverse transcriptase, or a combination thereof. In certain embodiments, the method comprises amplifying the plurality of barcoded control oligonucleotides to generate a plurality of amplicons. Amplifying the plurality of barcoded control oligonucleotides may comprise amplifying at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data may comprise obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data can include sequencing at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotide.

[0141] In some embodiments, a method for sequencing control may include contacting one or more cells of a plurality of cells with a control composition of a plurality of control compositions, wherein the one cell of the plurality of cells comprises a plurality of targets and a plurality of protein targets, each of the plurality of control compositions comprising a protein binding reagent associated with a control oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the plurality of protein targets, and the control oligonucleotide comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to the target binding region of at least one of the plurality of barcodes; and determining at least one characteristic of the one or more cells using at least one characteristic of the plurality of control oligonucleotides. The pseudo target region may comprise a poly(dA) region.

[0142] In some embodiments, the control barcode sequence is at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The control particle oligonucleotide can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. At least 2, 10, 100, 1000, or more of the control barcode sequences of the plurality of control particle oligonucleotides can be identical. At least 2, 10, 100, 1000, or more of the plurality of control particle oligonucleotides can contain different control barcode sequences.

[0143] In some embodiments, determining at least one characteristic of the one or more cells includes: determining the number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides in the sequencing data; and determining the number of one or more cells using the number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides. The method may include determining a single-cell capture efficiency based on the determined number of the one or more cells. The method may include determining a single-cell capture efficiency based on a ratio of the determined number of the one or more cells to the number of the plurality of cells.

[0144] In some embodiments, determining at least one characteristic of one or more cells using features of a plurality of barcoded control oligonucleotides in the sequencing data includes: determining, for each cell marker in the sequencing data, the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence; and determining the number of one or more cells using the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence. Determining the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence may include, for each cell marker in the sequencing data, determining the number of molecular marker sequences having the most distinguishable sequences associated with the cell marker and the control barcode sequence. Determining the number of one or more cells using the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence may include creating a plot of the number of cell markers and the number of molecular marker sequences having the most distinguishable sequences in the sequencing data associated with the number of molecular marker sequences having the most distinguishable sequences; and determining a cutoff in the plot as the number of one or more cells.

[0145] In some embodiments, the control oligonucleotide is not homologous to any genomic sequence of the plurality of cells. The control oligonucleotide can be homologous to a genomic sequence of one species. The species can be a non-mammalian species. The non-mammalian species can be a phage species. The phage species can be T7 phage, PhiX phage, or a combination thereof.

[0146] In some embodiments, the method includes releasing the control oligonucleotide from the protein-binding reagent before barcoding the control oligonucleotide. In some embodiments, the method includes removing unbound control compositions of the plurality of control compositions. Removing unbound control compositions may include washing one or more cells of the plurality of cells with a wash buffer. Removing unbound sample-indexing compositions may include using flow cytometry to select cells bound to at least one protein-binding reagent of the control composition.

[0147] In some embodiments, at least one of the multiple protein targets is on a cell surface. At least one of the multiple protein targets may include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The protein binding reagent may include an antibody. The control oligonucleotide may be attached to the protein binding reagent via a linker. The control oligonucleotide may include a linker. The linker may include a chemical group. The chemical group may be reversibly attached to the first protein binding reagent. The chemical group may include a UV photocleavable group, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0148] In some embodiments, the protein binding reagent is associated with two or more control oligonucleotides having the same control barcode sequence. The protein binding reagent may be associated with two or more control oligonucleotides having different identical control barcode sequences. In some embodiments, the second protein binding reagent of the plurality of control compositions is not associated with a control oligonucleotide. The protein binding reagent and the second protein binding reagent may be the same.

[0149] In some embodiments, the barcode comprises a binding site for a universal primer. The target binding region may comprise a poly(dT) region. In some embodiments, the plurality of barcodes is associated with a bar-coding particle. At least one barcode of the plurality of barcodes may be immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes may be partially immobilized on the bar-coding particle. At least one barcode of the plurality of barcodes is encapsulated in the bar-coding particle. At least one barcode of the plurality of barcodes is partially encapsulated in the bar-coding particle. The bar-coding particle may be disintegrable. The bar-coding particle may be a bar-coding bead. The bar-coding beads may include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The bar-coding particles may include polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof. The bar-coding particles may include degradable hydrogel particles.

[0150] In some embodiments, the bar-coded particle is associated with an optical moiety. The control oligonucleotide can be associated with an optical moiety.

[0151] In some embodiments, the method includes barcoding a control oligonucleotide using a plurality of barcodes to generate a plurality of barcoded control oligonucleotides, each of the plurality of barcodes comprising a cell label sequence, a molecular label sequence, and a target binding region, wherein the molecular label sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell label sequence; and obtaining sequencing data for the plurality of barcoded control oligonucleotides.

[0152] In some embodiments, the barcode of the barcode particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. In some embodiments, the molecular beacon sequence of the barcode comprises a random sequence. In some embodiments, the barcode particle comprises at least 10,000 barcodes.

[0153] In some embodiments, barcoding the control oligonucleotides comprises barcoding the control oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded control oligonucleotides. In some embodiments, barcoding the plurality of control oligonucleotides with a plurality of barcodes comprises contacting the plurality of barcodes with a plurality of control oligonucleotides of a control composition to generate barcodes hybridized to the control oligonucleotides; and extending the barcodes hybridized to the control oligonucleotides to generate a plurality of barcoded control oligonucleotides. Extending the barcodes may comprise extending the barcodes using a DNA polymerase, a reverse transcriptase, or a combination thereof. In some embodiments, the method comprises amplifying the plurality of barcoded control oligonucleotides to generate a plurality of amplicons. Amplifying the plurality of barcoded control oligonucleotides may comprise amplifying at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotides using polymerase chain reaction (PCR). In some embodiments, obtaining sequencing data comprises obtaining sequencing data for the plurality of amplicons. Obtaining sequencing data can include sequencing at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotide.

[0154] In some embodiments, a method for cell identification includes contacting a first plurality of cells and a second plurality of cells with two sample indexing compositions, respectively, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more antigen targets, each of the two sample indexing compositions comprising an antigen binding reagent associated with a sample indexing oligonucleotide, the antigen binding reagent capable of specifically binding to at least one of the one or more antigen targets, the sample indexing oligonucleotide comprising a sample indexing sequence, the sample indexing sequences of the two sample indexing compositions comprising different sequences; barcoding the sample indexing oligonucleotides using a plurality of barcodes to form a plurality of barcoded sample indexes. The method may include generating indexed oligonucleotides, wherein each of a plurality of barcodes comprises a cell label sequence, a barcode sequence (e.g., a molecular label sequence), and / or a target binding region, wherein the barcode sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and wherein at least two barcodes of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded sample-indexed oligonucleotides; identifying cell label sequences associated with two or more sample-indexed sequences in the obtained sequencing data; and removing the sequencing data associated with the cell label sequences from the obtained sequencing data and / or excluding the sequencing data associated with the cell label sequences from subsequent analysis. In some embodiments, the sample-indexed oligonucleotides comprise a barcode sequence, a binding site for a universal primer, or a combination thereof.

[0155] Disclosed herein also includes methods for identifying multiplets. A multiplet expression profile, or multiplet, can be an expression profile that includes the expression profiles of multiple cells. When determining the expression profile of a single cell, n cells may be identified as one cell, and the expression profile of n cells may be identified as the expression profile of one cell (referred to as a multiplet or n-plet expression profile). For example, when determining the expression profile of two cells using barcoding (e.g., probabilistic barcoding), mRNA molecules of the two cells may be associated with barcodes having the same cell label. As another example, two cells may be associated with one particle (e.g., a bead). The particle may contain the same cell label barcode. After lysing the cells, mRNA molecules in the two cells may be associated with the particle, and thus the same cell label barcode. Doublet expression profiles can skew the interpretation of the expression profile. Multiplets may be different in various implementations. In some embodiments, the plurality of multiplets may include doublets, triplets, quartets, quintets, sextets, septets, octets, nonets, or any combination thereof.

[0156] A multiplet can be any n-plet. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range between or about any two of these values. In some embodiments, n is at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. A singlet can be an expression profile that is not a multiplet expression profile.

[0157] The results of using sample-indexed oligonucleotides to index samples and identify multiplets can be comparable to the results of using expression profiles of synthetic multiplets to identify multiplets (as described in U.S. Patent Application No. 15 / 926,977, filed March 20, 2018, entitled "SYNTHETIC MULTIPLETS FOR MULTIPLETS DETERMINATION," the contents of which are incorporated herein by reference in their entirety). In some embodiments, multiplets can be identified using both sample-indexed oligonucleotides and expression profiles of synthetic multiplets.

[0158] In some embodiments, the method of identifying multiplets disclosed herein includes contacting a first plurality of cells and a second plurality of cells, respectively, with two sample indexing compositions, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more antigen targets, each of the two sample indexing compositions comprising an antigen binding reagent associated with a sample indexing oligonucleotide, the antigen binding reagent capable of specifically binding to at least one of the one or more antigen targets, the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprising different sequences; a target binding region, wherein the target binding region is a molecular label sequence, and the molecular label sequence of at least two barcodes of the plurality of barcodes comprises different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying one or more multicellular label sequences each associated with the two or more sample-indexing sequences in the obtained sequencing data. In some embodiments, the method includes removing sequencing data associated with one or more multicellular label sequences from the obtained sequencing data and / or excluding sequencing data associated with one or more multicellular label sequences from subsequent analysis. In some embodiments, the sample-indexing oligonucleotide comprises, for example, a molecular label sequence, a binding site for a universal primer, or a combination thereof.

[0159] In some embodiments, contacting the first plurality of cells and the second plurality of cells with each of the two sample indexing compositions comprises: contacting the first plurality of cells with a first sample indexing composition of the two sample indexing compositions; and contacting the first plurality of cells with a second sample indexing composition of the two sample indexing compositions.

[0160] In some embodiments, the sample indexing sequences are at least 6 nucleotides in length, 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The sample indexing oligonucleotides can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. In some embodiments, the sample indexing sequences of at least 10, 100, 1000, or more sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0161] In some embodiments, the antigen-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the antigen-binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the antigen-binding reagent. The chemical group may comprise a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0162] In some embodiments, at least one of the first plurality of cells and the second plurality of cells comprises a single cell. At least one of the one or more antigen targets can be on the cell surface.

[0163] In some embodiments, the method includes removing unbound sample indexing compositions of the two sample indexing compositions. Removing unbound sample indexing compositions may include washing the cells of the first plurality of cells and the second plurality of cells with a wash buffer. Removing unbound sample indexing compositions may include selecting cells bound to at least one antigen-binding reagent of the two sample indexing compositions using flow cytometry. In some embodiments, the method includes lysing one or more cells of the first plurality of cells and the second plurality of cells.

[0164] In some embodiments, the sample index-added oligonucleotide is configured to be cleavable or non-cleavable from the antigen-binding reagent. The method may include cleaving the sample index-added oligonucleotide from the antigen-binding reagent. Cleavage of the sample index-added oligonucleotide may include cleaving the sample index-added oligonucleotide from the antigen-binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0165] In some embodiments, the sample-indexing oligonucleotide is not homologous to any genomic sequence of one or more cells. The control barcode sequence may not be homologous to the genomic sequence of a species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species may be T7 phage, PhiX phage, or a combination thereof.

[0166] In some embodiments, the first plurality of cells and the second plurality of cells comprise tumor cells, mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes. The barcode may comprise a target binding region comprising the capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(dA) region.

[0167] In some embodiments, the antigen target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The antigen target may be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The antigen target may be or include a lipid, a carbohydrate, or any combination thereof. The antigen target may be selected from a group comprising 10 to 100 different antigen targets.

[0168] In some embodiments, an antigen-binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence. An antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences. A sample-indexing composition of the plurality of sample-indexing compositions may include a second antigen-binding reagent that is not associated with a sample-indexing oligonucleotide. The antigen-binding reagent and the second antigen-binding reagent may be the same.

[0169] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences. The barcode may comprise a cell beacon sequence, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0170] In some embodiments, a plurality of barcodes can be associated with a particle. At least one barcode of the plurality of barcodes can be immobilized on a particle. At least one barcode of the plurality of barcodes can be partially immobilized on a particle. At least one barcode of the plurality of barcodes can be encapsulated in a particle. At least one barcode of the plurality of barcodes can be partially encapsulated in a particle. The particle can be disintegrable. The particle can be a bead. The bead can be or include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane / (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise collapsible hydrogel particles.

[0171] In some embodiments, the antigen-binding reagent is associated with a detectable moiety. In some embodiments, the particle is associated with a detectable moiety. The sample-indexing oligonucleotide is associated with an optical moiety. In some embodiments, the barcode of the particle can comprise a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. The molecular beacon sequence of the barcode can comprise a random sequence. The particle can comprise at least 10,000 barcodes.

[0172] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0173] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of a molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). In some embodiments, acquiring sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include acquiring sequencing data for the plurality of amplicons. Acquiring the sequencing data includes sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides. In some embodiments, identifying the sample origin of the at least one cell includes identifying the sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded, sample-indexing oligonucleotide.

[0174] In some embodiments, barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

[0175] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; and obtaining sequencing data for the barcoded targets. Barcoding the plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets can include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets.

[0176] In some embodiments, the method includes amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding the plurality of cellular targets with the plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding the plurality of cellular targets with the plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0177] In some embodiments, a method for cell identification includes contacting a first plurality of cells and a second plurality of cells, respectively, with two sample-indexing compositions, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets, each of the two sample-indexing compositions comprising a cellular component binding reagent associated with a sample-indexing oligonucleotide, wherein the cellular component binding reagent is capable of specifically binding to at least one of the one or more cellular component targets, the sample-indexing oligonucleotide comprising a sample-indexing sequence, wherein the sample-indexing sequences of two of the plurality of sample-indexing compositions comprise different sequences; barcoding the sample-indexing oligonucleotides using a plurality of barcodes; and The method may include generating a plurality of barcodes, each of which comprises a cell label sequence, a molecular label sequence, and a target binding region, wherein the molecular label sequences of at least two of the plurality of barcodes comprise different sequences, and at least two of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded sample-indexing oligonucleotides; identifying one or more cell label sequences each associated with two or more sample-indexing sequences in the obtained sequencing data; removing from the obtained sequencing data the sequencing data associated with the one or more cell label sequences each associated with the two or more sample-indexing sequences and / or excluding from subsequent analysis the sequencing data associated with the one or more cell label sequences each associated with the two or more sample-indexing sequences. In some embodiments, the sample-indexing oligonucleotide comprises a molecular label sequence, a binding site for a universal primer, or a combination thereof.

[0178] Disclosed herein are methods for identifying multiplets. In some embodiments, the methods include contacting a first plurality of cells and a second plurality of cells, respectively, with two sample-indexing compositions, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets, each of the two sample-indexing compositions comprising a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets, the sample-indexing oligonucleotide comprising a sample-indexing sequence, and the sample-indexing sequences of two of the plurality of sample-indexing compositions comprising different sequences; The method includes: barcoding sample-indexing oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides, each of the plurality of barcodes comprising a cell label sequence, a molecular label sequence, and a target-binding region, wherein the molecular label sequences of at least two of the plurality of barcodes comprise different sequences, and at least two of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying one or more multicellular label sequences each associated with the two or more sample-indexing sequences in the obtained sequencing data. In some embodiments, the method includes removing sequencing data associated with one or more multicellular label sequences from the obtained sequencing data and / or excluding sequencing data associated with one or more multicellular label sequences from subsequent analysis. In some embodiments, the sample-indexing oligonucleotide comprises a molecular label sequence, a binding site for a universal primer, or a combination thereof.

[0179] In some embodiments, contacting the first plurality of cells and the second plurality of cells with two sample indexing compositions, respectively, comprises: contacting the first plurality of cells with a first sample indexing composition of the two sample indexing compositions; and contacting the first plurality of cells with a second sample indexing composition of the two sample indexing compositions.

[0180] In some embodiments, the sample indexing sequences are at least 6 nucleotides in length, 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The sample indexing oligonucleotides can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. In some embodiments, the sample indexing sequences of at least 10, 100, 1000, or more sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0181] In some embodiments, the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the cellular component binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the cellular component binding reagent. The chemical group may comprise a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0182] In some embodiments, at least one of the first plurality of cells and the second plurality of cells comprises a single cell. At least one of the one or more cellular component targets can be on a cell surface.

[0183] In some embodiments, the method includes removing unbound sample indexing compositions of the two sample indexing compositions. Removing unbound sample indexing compositions may include washing the first plurality of cells and the second plurality of cells with a wash buffer. Removing unbound sample indexing compositions may include using flow cytometry to select cells bound to at least one cellular component binding reagent of the two sample indexing compositions. In some embodiments, the method includes lysing one or more cells of the first plurality of cells and the second plurality of cells.

[0184] In some embodiments, the sample-indexing oligonucleotide is configured to be cleavable or non-cleavable from the cellular component binding reagent. The method may include cleaving the sample-indexing oligonucleotide from the cellular component binding reagent. Cleavage of the sample-indexing oligonucleotide may include cleaving the sample-indexing oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment (e.g., with a reducing reagent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0185] In some embodiments, the sample-indexing oligonucleotide is not homologous to any genomic sequence of one or more cells. The control barcode sequence may not be homologous to the genomic sequence of a species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species is T7 phage, PhiX phage, or a combination thereof.

[0186] In some embodiments, the first plurality of cells and the second plurality of cells comprise tumor cells, mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes. The barcode may comprise a target binding region comprising the capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(dA) region.

[0187] In some embodiments, the antigen target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The antigen target may be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The antigen target may be or include a lipid, a carbohydrate, or any combination thereof. The antigen target may be selected from a group comprising 10 to 100 different antigen targets.

[0188] In some embodiments, a cellular component binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence. A cellular component binding reagent may be associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences. A sample-indexing composition of the plurality of sample-indexing compositions may include a second cellular component binding reagent that is not associated with a sample-indexing oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent may be the same.

[0189] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences. The barcode may comprise a cell beacon sequence, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0190] In some embodiments, a plurality of barcodes can be associated with a particle. At least one barcode of the plurality of barcodes can be immobilized on a particle. At least one barcode of the plurality of barcodes can be partially immobilized on a particle. At least one barcode of the plurality of barcodes can be encapsulated in a particle. At least one barcode of the plurality of barcodes can be partially encapsulated in a particle. The particle can be disintegrable. The particle can be a bead. The bead can be or include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise collapsible hydrogel particles.

[0191] In some embodiments, the cellular component binding reagent is associated with a detectable moiety. In some embodiments, the particle is associated with a detectable moiety. The sample-indexing oligonucleotide is associated with an optical moiety.

[0192] In some embodiments, the barcode of a particle can comprise a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. The molecular beacon sequence of the barcode can comprise a random sequence. The particle can comprise at least 10,000 barcodes.

[0193] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0194] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of a molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). In some embodiments, acquiring sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include acquiring sequencing data for the plurality of amplicons. Acquiring the sequencing data includes sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides. In some embodiments, identifying the sample origin of the at least one cell includes identifying the sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded, sample-indexing oligonucleotide.

[0195] In some embodiments, barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

[0196] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; and obtaining sequencing data for the barcoded targets. Barcoding the plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets can include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets.

[0197] In some embodiments, the method includes amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding the plurality of cellular targets with the plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding the plurality of cellular targets with the plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0198] Disclosed herein is a method for cell identification. In some embodiments, the method includes contacting one or more cells from each of a first plurality of cells and a second plurality of cells with a plurality of two sample indexing compositions, respectively, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more antigen targets, each of the two sample indexing compositions comprises an antigen binding reagent associated with a sample indexing oligonucleotide, the antigen binding reagent capable of specifically binding to at least one of the one or more antigen targets, the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; and identifying one or more cells each associated with the two or more sample indexing sequences. In some embodiments, the sample indexing oligonucleotide comprises a molecular beacon sequence, a binding site for a universal primer, or a combination thereof.

[0199] Disclosed herein are methods for identifying multiplets. In some embodiments, the methods include contacting one or more cells from each of a first plurality of cells and a second plurality of cells with a plurality of two sample-indexing compositions, respectively, wherein each of the first plurality of cells and each of the second plurality of cells comprises one or more antigen targets, each of the two sample-indexing compositions comprises an antigen-binding reagent associated with a sample-indexing oligonucleotide, the antigen-binding reagent capable of specifically binding to at least one of the one or more antigen targets, the sample-indexing oligonucleotide comprises a sample-indexing sequence, and the sample-indexing sequences of the two sample-indexing compositions comprise different sequences; and identifying one or more cells each associated with the two or more sample-indexing sequences as multiplets.

[0200] In some embodiments, identifying cells each associated with two or more sample-indexing sequences includes barcoding sample-indexing oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides, each barcode comprising a cell label sequence, a molecular label sequence, and a target-binding region, wherein the molecular label sequences of at least two of the plurality of barcodes comprise different sequences, and at least two of the plurality of barcodes comprise the same cell label sequence; acquiring sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying one or more cell label sequences each associated with the two or more sample-indexing sequences in the obtained sequencing data. The method may include removing sequencing data associated with one or more cell label sequences each associated with the two or more sample-indexing sequences from the obtained sequencing data and / or excluding from subsequent analysis the sequencing data associated with one or more cell label sequences each associated with the two or more sample-indexing sequences.

[0201] In some embodiments, contacting the first plurality of cells and the second plurality of cells with two sample indexing compositions, respectively, comprises: contacting the first plurality of cells with a first sample indexing composition of the two sample indexing compositions; and contacting the first plurality of cells with a second sample indexing composition of the two sample indexing compositions.

[0202] In some embodiments, the sample indexing sequences are at least 6 nucleotides in length, 25-60 nucleotides in length (e.g., 45 nucleotides in length), about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, or a combination thereof. The sample indexing oligonucleotides can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or a combination thereof. In some embodiments, the sample indexing sequences of at least 10, 100, 1000, or more sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0203] In some embodiments, the antigen-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the antigen-binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the antigen-binding reagent. The chemical group may comprise a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0204] In some embodiments, at least one of the first plurality of cells and the second plurality of cells comprises a single cell. At least one of the one or more antigen targets may be on a cell surface. In some embodiments, the method includes removing unbound sample indexing compositions of the two sample indexing compositions. Removing unbound sample indexing compositions may include washing the first plurality of cells and the second plurality of cells with a wash buffer. Removing unbound sample indexing compositions may include using flow cytometry to select cells bound to at least one antigen-binding reagent of the two sample indexing compositions. In some embodiments, the method includes lysing one or more cells of the first plurality of cells and the second plurality of cells.

[0205] In some embodiments, the sample index-added oligonucleotide is configured to be cleavable or non-cleavable from the antigen-binding reagent. The method may include cleaving the sample index-added oligonucleotide from the antigen-binding reagent. Cleavage of the sample index-added oligonucleotide may include cleaving the sample index-added oligonucleotide from the antigen-binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0206] In some embodiments, the sample-indexing oligonucleotide is not homologous to any genomic sequence of one or more cells. The control barcode sequence may not be homologous to the genomic sequence of a species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species may be T7 phage, PhiX phage, or a combination thereof.

[0207] In some embodiments, the first plurality of cells and the second plurality of cells comprise tumor cells, mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes. The barcode may comprise a target binding region comprising the capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(dA) region.

[0208] In some embodiments, the antigen target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The antigen target can be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The antigen target can be or include a lipid, a carbohydrate, or any combination thereof. The antigen target can be selected from a group comprising 10 to 100 different antigen targets.

[0209] In some embodiments, an antigen-binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence. An antigen-binding reagent may be associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences. A sample-indexing composition of the plurality of sample-indexing compositions may include a second antigen-binding reagent that is not associated with a sample-indexing oligonucleotide. The antigen-binding reagent and the second antigen-binding reagent may be the same.

[0210] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences. The barcode may comprise a cell beacon sequence, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0211] In some embodiments, a plurality of barcodes can be associated with a particle. At least one barcode of the plurality of barcodes can be immobilized on a particle. At least one barcode of the plurality of barcodes can be partially immobilized on a particle. At least one barcode of the plurality of barcodes can be encapsulated in a particle. At least one barcode of the plurality of barcodes can be partially encapsulated in a particle. The particle can be disintegrable. The particle can be a bead. The bead can be or include sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise collapsible hydrogel particles.

[0212] In some embodiments, the antigen-binding reagent is associated with a detectable moiety. In some embodiments, the particle is associated with a detectable moiety. The sample-indexing oligonucleotide is associated with an optical moiety.

[0213] In some embodiments, the barcode of a particle can comprise a molecular beacon sequence selected from at least 1,000, 10,000, or more different molecular beacon sequences. The molecular beacon sequence of the barcode can comprise a random sequence. The particle can comprise at least 10,000 barcodes.

[0214] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0215] In some embodiments, the method includes amplifying a plurality of barcoded, sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded, sample-indexing oligonucleotides may include amplifying at least a portion of a molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). In some embodiments, acquiring sequencing data for the plurality of barcoded, sample-indexing oligonucleotides may include acquiring sequencing data for the plurality of amplicons. Acquiring the sequencing data includes sequencing at least a portion of the barcode sequence and at least a portion of the sample-indexing oligonucleotides. In some embodiments, identifying the sample origin of the at least one cell includes identifying the sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded, sample-indexing oligonucleotide.

[0216] In some embodiments, barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

[0217] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; and obtaining sequencing data for the barcoded targets. Barcoding the plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets can include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets.

[0218] In some embodiments, the method includes amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding the plurality of cellular targets with the plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding the plurality of cellular targets with the plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0219] Disclosed herein are systems, methods, and kits for determining interactions between cellular component targets, e.g., interactions between proteins. In some embodiments, the methods include contacting a cell with a first pair of interaction determining compositions, wherein the cell comprises a first protein target and a second protein target, each of the first pair of interaction determining compositions comprising a protein binding reagent associated with an interaction determining oligonucleotide, wherein one protein binding reagent of the first pair of interaction determining compositions is capable of specifically binding to the first protein target and the other protein binding reagent of the first pair of interaction determining compositions is capable of specifically binding to the second protein target, the interaction determining oligonucleotide comprises an interaction determining sequence and a bridging oligonucleotide hybridization region, wherein the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; The method includes: ligating the bridging oligonucleotides to generate ligated interaction determination oligonucleotides, wherein the bridging oligonucleotide comprises two hybridization regions capable of specifically binding to the hybridization regions of a first pair of bridging oligonucleotides of the interaction determination composition; barcoding the ligated interaction determination oligonucleotides using a plurality of barcodes to generate a plurality of barcoded interaction determination oligonucleotides, wherein each of the plurality of barcodes comprises a barcode sequence and a capture sequence; obtaining sequencing data of the plurality of barcoded interaction determination oligonucleotides; and determining the interaction between the first and second protein targets based on the association of the interaction determination sequences of the first pair of interaction determination compositions in the obtained sequencing data.

[0220] Disclosed herein are systems, methods, and kits for determining interactions between cellular component targets. In some embodiments, the method includes contacting a cell with a first pair of interaction determining compositions, wherein the cell comprises a first cellular component target and a second cellular component target, each of the first pair of interaction determining compositions comprising a cellular component binding reagent associated with an interaction determining oligonucleotide, wherein one cellular component binding reagent of the first pair of interaction determining compositions is capable of specifically binding to the first cellular component target and the other cellular component binding reagent of the first pair of interaction determining compositions is capable of specifically binding to the second cellular component target, the interaction determining oligonucleotide comprises an interaction determining sequence and a bridging oligonucleotide hybridization region, and the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; and using the bridging oligonucleotide to bind the interaction determining oligonucleotides of the first pair of interaction determining compositions. The method includes: ligating the ligated interaction determination oligonucleotides to generate ligated interaction determination oligonucleotides, wherein the bridging oligonucleotide comprises two hybridization regions capable of specifically binding to the hybridization regions of a first pair of bridging oligonucleotides of the interaction determination composition; barcoding the ligated interaction determination oligonucleotides using a plurality of barcodes to generate a plurality of barcoded interaction determination oligonucleotides, wherein each of the plurality of barcodes comprises a barcode sequence and a capture sequence; obtaining sequencing data for the plurality of barcoded interaction determination oligonucleotides; and determining the interaction between the first and second cellular component targets based on the association of the first pair of interaction determination sequences of the interaction determination composition in the obtained sequencing data. At least one of the two cellular component binding reagents may comprise a protein binding reagent. The protein binding reagent may be associated with one of the two interaction determination oligonucleotides. The one or more cellular component targets may comprise at least one protein target.

[0221] In some embodiments, contacting the cell with the first pair of interaction determining compositions comprises sequentially or simultaneously contacting the cell with each of the first pair of interaction determining compositions. The first protein target can be the same as the second protein target, or the first protein target can be different from the second protein target.

[0222] The length of the interaction determining sequence can vary. For example, the interaction determining sequence can be from 2 nucleotides to about 1000 nucleotides in length. In some embodiments, the interaction determining sequence can be at least 6 nucleotides in length, 25 to 60 nucleotides in length, about 45 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, about 200 to 300 nucleotides in length, about 200 to 500 nucleotides in length, about 500 nucleotides in length, or any combination thereof.

[0223] In some embodiments, the method includes contacting the cell with a second pair of interaction determining compositions, wherein the cell comprises a third protein target and a fourth protein target, each of the second pair of interaction determining compositions comprising a protein binding reagent associated with an interaction determining oligonucleotide, wherein one protein binding reagent of the second pair of interaction determining compositions is capable of specifically binding to the third protein target and the other protein binding reagent of the second pair of interaction determining compositions is capable of specifically binding to the fourth protein target. At least one of the third and fourth protein targets can be different from one of the first and second protein targets. In some embodiments, at least one of the third and fourth protein targets and at least one of the first and second protein targets can be identical.

[0224] In some embodiments, the method includes contacting a cell with three or more pairs of interaction determining compositions. The interaction determining sequences of at least 10 interaction determining compositions of the plurality of pairs of interaction determining compositions may comprise different sequences. The interaction determining sequences of at least 100 interaction determining compositions of the plurality of pairs of interaction determining compositions may comprise different sequences. The interaction determining sequences of at least 1000 interaction determining compositions of the plurality of pairs of interaction determining compositions may comprise different sequences.

[0225] In some embodiments, the hybridization regions of the first pair of bridging oligonucleotides of the interaction determining composition comprise different sequences, and at least one of the bridging oligonucleotide hybridization regions can be complementary to at least one of the two hybridization regions of the bridging oligonucleotide.

[0226] In some embodiments, the step of ligating a first pair of interaction determining oligonucleotides of the interaction determining composition using a bridging oligonucleotide comprises: hybridizing a first hybridization region of the bridging oligonucleotide to a first bridging oligonucleotide hybridization region of the bridging oligonucleotide hybridization region of the interaction determining oligonucleotide; hybridizing a second hybridization region of the bridging oligonucleotide to a second bridging oligonucleotide hybridization region of the bridging oligonucleotide hybridization region of the interaction determining oligonucleotide; and ligating the interaction determining oligonucleotides hybridized to the bridging oligonucleotides to produce ligated interaction determining oligonucleotides.

[0227] In some embodiments, the protein-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, an integrin, or a combination thereof. The interaction-determining oligonucleotide may be attached to the protein-binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the protein-binding reagent. The chemical group may comprise a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof.

[0228] The location of a protein target in a cell can vary, for example, depending on whether it is on the cell surface or inside the cell. In some embodiments, at least one of the one or more protein targets is on the cell surface. In some embodiments, at least one of the one or more protein targets is an intracellular protein. In some embodiments, at least one of the one or more protein targets is a transmembrane protein. In some embodiments, at least one of the one or more protein targets is an extracellular protein. In some embodiments, the method can include fixing the cells before contacting the cells with the first pair of interaction determining compositions. In some embodiments, the method can include removing unbound interaction determining compositions of the first pair of interaction determining compositions. Removing unbound interaction determining compositions can include washing the cells with a wash buffer. Removing unbound interaction determining compositions can include selecting the cells using flow cytometry. In some embodiments, the method can include lysing the cells.

[0229] In some embodiments, the interaction-determining oligonucleotide is configured to be detachable or non-detachable from the protein-binding reagent. The method may include detaching the interaction-determining oligonucleotide from the protein-binding reagent. The detaching step may include detaching the interaction-determining oligonucleotide from the protein-binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof. The interaction-determining oligonucleotide may not be homologous to a genomic sequence of a cell. The interaction-determining oligonucleotide may be homologous to a genomic sequence of a species. The species may be a non-mammalian species. The non-mammalian species may be a phage species. The phage species may be T7 phage, PhiX phage, or a combination thereof. In some embodiments, one interaction-determining oligonucleotide of the first pair of the interaction-determining composition comprises a sequence complementary to a capture sequence. The capture sequence may comprise a poly(dT) region. The sequence of the interaction-determining oligonucleotide complementary to the capture sequence may comprise a poly(dA) region. In some embodiments, the interaction-determining oligonucleotide comprises a second barcode sequence. The other interaction-determining oligonucleotide of the first pair of interaction identification compositions may comprise a binding site for a universal primer. The interaction-determining oligonucleotide may be associated with a detectable moiety.

[0230] The protein-binding reagent can be associated with two or more interaction-determining oligonucleotides having different interaction-determining sequences. In some embodiments, one of the plurality of interaction-determining compositions includes a second protein-binding reagent that is not associated with an interaction-determining oligonucleotide. The protein-binding reagent and the second protein-binding reagent can be identical. The protein-binding reagent can be associated with a detectable moiety.

[0231] In some embodiments, the cells are tumor cells or non-tumor cells. For example, the cells can be mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof. In some embodiments, the method includes contacting two or more cells with a first pair of interaction determining compositions, wherein each of the two or more cells comprises a first and a second protein target. At least one of the two or more cells can comprise a single cell.

[0232] In some embodiments, the protein target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The protein target may be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The protein target may be or include a lipid, a carbohydrate, or any combination thereof. The protein target may be selected from a group comprising 10 to 100 different protein targets. The protein binding reagent may be associated with two or more interaction-determining oligonucleotides having the same sequence.

[0233] In some embodiments, the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof. At least two barcodes of the plurality of barcodes may comprise the same cell labeling sequence. In some embodiments, the plurality of barcodes are associated with a particle. At least one barcode of the plurality of barcodes may be immobilized on a particle. At least one barcode of the plurality of barcodes may be partially immobilized on a particle. At least one barcode of the plurality of barcodes may be encapsulated in a particle. At least one barcode of the plurality of barcodes may be partially encapsulated in a particle. The particle may be disintegrable. The particle may comprise a bead. The particles may include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may include collapsible hydrogel particles. The particles may be associated with a detectable moiety.

[0234] In some embodiments, the barcode of the particle comprises a barcode sequence selected from at least 1,000 different barcode sequences. The barcode of the particle may comprise a barcode sequence selected from at least 10,000 different barcode sequences. The barcode sequence of the barcode may comprise a random sequence. The particle may comprise at least 10,000 barcodes.

[0235] In some embodiments, barcoding the interaction-determining oligonucleotides using a plurality of barcodes includes contacting the plurality of barcodes with the interaction-determining oligonucleotides to generate barcodes hybridized to the interaction-determining oligonucleotides; and extending the barcodes hybridized to the interaction-determining oligonucleotides to generate a plurality of barcoded interaction-determining oligonucleotides. Extending the barcodes may include extending the barcodes using a DNA polymerase to generate a plurality of barcoded interaction-determining oligonucleotides. Extending the barcodes may include extending the barcodes using a reverse transcriptase to generate a plurality of barcoded interaction-determining oligonucleotides. Extending the barcodes may include extending the barcodes using Moloney murine leukemia virus (M-MLV) reverse transcriptase or Taq DNA polymerase to generate a plurality of barcoded interaction-determining oligonucleotides. Extending the barcodes may include displacing a bridging oligonucleotide from the ligated interaction-determining oligonucleotides.

[0236] In some embodiments, the method includes amplifying a plurality of barcoded interaction determination oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded interaction determination oligonucleotides may include amplifying at least a portion of the barcode sequences and at least a portion of the interaction determination oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data of the plurality of barcoded interaction determination oligonucleotides may include obtaining sequencing data of the plurality of amplicons. Obtaining sequencing data may include sequencing at least a portion of the barcode sequences and at least a portion of the interaction determination oligonucleotides.

[0237] In some embodiments, obtaining sequencing data for the plurality of barcoded interaction determining oligonucleotides comprises obtaining partial and / or complete sequences of the plurality of barcoded interaction determining oligonucleotides.

[0238] In some embodiments, the plurality of barcodes comprises a plurality of probabilistic barcodes. The barcode sequence of each of the plurality of probabilistic barcodes may comprise a molecular beacon sequence. The molecular beacon sequences of at least two of the plurality of probabilistic barcodes may comprise different sequences. Barcoding the interaction-determining oligonucleotides using the plurality of barcodes to generate a plurality of barcoded interaction-determining oligonucleotides may comprise barcoding the interaction-determining oligonucleotides using the plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded interaction-determining oligonucleotides.

[0239] In some embodiments, the method includes barcoding a plurality of targets on cells with a plurality of barcodes to generate a plurality of barcoded targets; and acquiring sequencing data for the barcoded targets. Barcoding a plurality of targets on cells with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. The method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets before acquiring sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding a plurality of targets on cells with a plurality of barcodes to generate a plurality of barcoded targets may include probabilistic barcoding the plurality of targets on cells with a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0240] Disclosed herein is a kit for identifying interactions between cellular components, for example, protein-protein interactions. In some embodiments, the kit includes a first pair of interaction determining compositions, each of which comprises a protein-binding reagent associated with an interaction determining oligonucleotide, wherein one protein-binding reagent of the first pair of interaction determining compositions is capable of specifically binding to a first protein target and the other protein-binding reagent of the first pair of interaction determining compositions is capable of specifically binding to a second protein target, the interaction determining oligonucleotides comprise an interaction determining sequence and a bridging oligonucleotide hybridization region, and the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; and a plurality of bridging oligonucleotides, each of which comprises two hybridization regions capable of specifically binding to the bridging oligonucleotide hybridization regions of the first pair of interaction determining compositions.

[0241] The length of the interaction determining sequence can vary, in some embodiments, the interaction determining sequence is at least 6 nucleotides in length, 25-60 nucleotides in length, about 45 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 128 nucleotides in length, at least 128 nucleotides in length, about 200-500 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 500 nucleotides in length, or any combination thereof.

[0242] In some embodiments, the kit further includes a second pair of interaction determining compositions, each of which comprises a protein binding reagent associated with an interaction determining oligonucleotide, wherein one protein binding reagent of the second pair of interaction determining compositions is capable of specifically binding to a third protein target and the other protein binding reagent of the second pair of interaction determining compositions is capable of specifically binding to a fourth protein target. At least one of the third and fourth protein targets can be different from one of the first and second protein targets. At least one of the third and fourth protein targets and at least one of the first and second protein targets can be identical.

[0243] In some embodiments, the kit may include three or more pairs of interaction determining compositions. The interaction determining sequences of at least 10 interaction determining compositions of the three or more pairs of interaction determining compositions may comprise different sequences. The interaction determining sequences of at least 100 interaction determining compositions of the three or more pairs of interaction determining compositions may comprise different sequences. The interaction determining sequences of at least 1000 interaction determining compositions of the three or more pairs of interaction determining compositions may comprise different sequences.

[0244] In some embodiments, the bridging oligonucleotide hybridization regions of two interaction determining compositions of the plurality of interaction determining compositions comprise different sequences, and at least one of the bridging oligonucleotide hybridization regions can be complementary to at least one of the two hybridization regions of the bridging oligonucleotide.

[0245] In some embodiments, the protein-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The interaction-determining oligonucleotide may be attached to the protein-binding reagent via a linker. At least one interaction-determining oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the protein-binding reagent. The chemical group may comprise a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, a disulfide bond, or any combination thereof. The interaction-determining oligonucleotide is not homologous to the genomic sequence of any cell of interest. The cell of interest may comprise a tumor cell or a non-tumor cell. The cell of interest may be a single cell, a mammalian cell, a bacterial cell, a viral cell, a yeast cell, a fungal cell, or any combination thereof.

[0246] In some embodiments, the kit further includes a plurality of barcodes, each of the plurality of barcodes comprising a barcode sequence and a capture sequence. One interaction-determining oligonucleotide of the first pair of the interaction-determining composition may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The capture sequence may comprise a poly(dT) region. The sequence of the interaction-determining oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(dA) region. The other interaction-determining oligonucleotide of the first pair of the interaction identification composition may comprise a cell label sequence, a binding site for a universal primer, or any combination thereof. The plurality of barcodes may include a plurality of probabilistic barcodes, each barcode sequence of the plurality of probabilistic barcodes comprising a molecular label sequence, and the molecular label sequences of at least two of the plurality of probabilistic barcodes comprise different sequences.

[0247] In some embodiments, the protein target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The protein target can be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, or any combination thereof. The protein target can be selected from a group comprising 10 to 100 different protein targets. In some embodiments, the protein binding reagent can be associated with two or more interaction-determining oligonucleotides having the same sequence. The protein binding reagent can be associated with two or more interaction-determining oligonucleotides having different interaction-determining sequences. In some embodiments, the protein binding reagent can be associated with a detectable moiety.

[0248] In some embodiments, one of the first pair of interaction-determining compositions includes a second protein-binding reagent that is not associated with the interaction-determining oligonucleotide. The first protein-binding reagent and the second protein-binding reagent can be the same. The interaction-determining oligonucleotide can be associated with a detectable moiety.

[0249] In some embodiments, multiple barcodes are associated with a particle. At least one barcode of the multiple barcodes may be immobilized on a particle. At least one barcode of the multiple barcodes may be partially immobilized on a particle. At least one barcode of the multiple barcodes may be encapsulated in a particle. At least one barcode of the multiple barcodes may be partially encapsulated in a particle. The particle may be disintegrable. The particle may comprise beads. The particle may comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise collapsible hydrogel particles. The particles may be associated with a detectable moiety.

[0250] In some embodiments, the barcode of the particle comprises a barcode sequence selected from at least 1,000 different barcode sequences. The barcode of the particle may comprise a barcode sequence selected from at least 10,000 different barcode sequences. The barcode sequence of the barcode may comprise a random sequence. The particle may comprise at least 10,000 barcodes.

[0251] In some embodiments, the kit comprises a DNA polymerase, a reverse transcriptase, Moloney murine leukemia virus (M-MLV) reverse transcriptase, Taq DNA polymerase, or any combination thereof. The kit may comprise a fixative.

[0252] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with a sample indexing composition of a plurality of sample indexing compositions, wherein the one or more cells comprise one or more cellular component targets, each of the plurality of sample indexing compositions comprising a cellular component binding reagent (e.g., an antibody) associated with a sample indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets (e.g., a protein), the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; and identifying the samples using the plurality of barcodes. The method includes barcoding pull-index oligonucleotides to generate a plurality of barcoded sample-indexed oligonucleotides; amplifying the plurality of barcoded sample-indexed oligonucleotides with a plurality of daisy-chained amplification primers to generate a plurality of daisy-chained extension amplicons; obtaining sequencing data of the plurality of daisy-chained extension amplicons, the sequencing data of the plurality of barcoded sample-indexed oligonucleotides; and identifying a sample origin of at least one cell of the one or more cells based on the sample-indexed sequence of at least one barcoded sample-indexed oligonucleotide of the plurality of barcoded sample-indexed oligonucleotides. The method may include removing unbound sample-indexed compositions from the plurality of sample-indexed compositions.

[0253] In some embodiments, the sample indexing sequences are at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, or at least 128 nucleotides in length, or a combination thereof. The sample indexing oligonucleotides can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 200-500 nucleotides in length, about 500 nucleotides in length, or a combination thereof. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions can comprise different sequences. The sample indexing sequences of at least 100 or 1000 sample indexing compositions of the plurality of sample indexing compositions can comprise different sequences.

[0254] In some embodiments, the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the cellular component binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the cellular component binding reagent. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0255] In some embodiments, at least one sample of the plurality of samples comprises a single cell. At least one of the one or more cellular component targets may be on a cell surface. One sample of the plurality of samples may comprise a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0256] In some embodiments, removing unbound sample-indexing composition comprises washing one or more cells from each of the plurality of samples with a wash buffer. The method may comprise lysing one or more cells from each of the plurality of samples. The sample-indexing oligonucleotide may be configured to be detachable or non-detachable from the cellular component binding reagent. The method may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent. The detaching the sample-indexing oligonucleotide may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0257] In some embodiments, the sample indexing oligonucleotide is not homologous to the genomic sequence of cells of the plurality of samples. The sample indexing oligonucleotide may comprise a molecular label sequence, a poly(A) region, or a combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(A) tail. The sample indexing oligonucleotide may comprise a molecular label.

[0258] In some embodiments, the cellular component target is or includes an extracellular protein, an intracellular protein, or any combination thereof. The cellular component target may be or include a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof. The cellular component target may be or include a lipid, a carbohydrate, or any combination thereof. The cellular component target may be selected from a group including 10 to 100 different cellular component targets. The cellular component binding reagent may be associated with two or more sample indexing oligonucleotides having the same sequence. The cellular component binding reagent may be associated with two or more sample indexing oligonucleotides having different sample indexing sequences. The sample indexing composition of the multiple sample indexing compositions may include a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent may be identical.

[0259] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence. The molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences. The barcodes may comprise a cell beacon, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0260] In some embodiments, the plurality of barcodes are associated with a particle. At least one barcode of the plurality of barcodes can be immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof. The particle can be degradable. The particle can be a bead. The bead can be selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise at least 10,000 barcodes. In some embodiments, the barcodes of the particles may comprise molecular beacon sequences selected from at least 1,000 or 10,000 different molecular beacon sequences. The molecular beacon sequences of the barcodes may comprise random sequences.

[0261] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0262] In some embodiments, amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying the plurality of barcoded sample-indexing oligonucleotides using polymerase chain reaction (PCR). Amplifying the plurality of barcoded sample-indexing oligonucleotides may comprise amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides. Acquiring sequencing data for the plurality of barcoded sample-indexing oligonucleotides may comprise acquiring sequencing data for a plurality of daisy-chained extension amplicons. Acquiring sequencing data may comprise sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

[0263] In some embodiments, each daisy-chained amplification primer of the plurality of daisy-chained amplification primers comprises a barcoded sample index-added oligonucleotide binding region and an overhang region, and the barcoded sample index-added oligonucleotide binding region is capable of binding to the daisy-chained sample index-added region of the sample index-added oligonucleotide. The barcoded sample index-added oligonucleotide binding region may be at least 20 nucleotides in length, at least 30 nucleotides in length, about 40 nucleotides in length, at least 40 nucleotides in length, about 50 nucleotides in length, or a combination thereof. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise barcoded sample index-added oligonucleotide binding regions having the same sequence. The plurality of daisy-chained amplification primers may comprise barcoded sample index-added oligonucleotide binding regions having the same sequence. The overhang region may be at least 50 nucleotides in length, at least 100 nucleotides in length, at least 150 nucleotides in length, about 150 nucleotides in length, at least 200 nucleotides in length, or a combination thereof. The overhang region may comprise a daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having the same daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having two daisy-chained amplification primer barcode sequences. The overhang regions of the plurality of daisy-chained amplification primers may comprise different daisy-chained amplification primer barcode sequences.The daisy-chained extension amplicons of the plurality of daisy-chained extension amplicons can be at least 250 nucleotides in length, at least 300 nucleotides in length, at least 350 nucleotides in length, at least 400 nucleotides in length, about 400 nucleotides in length, at least 450 nucleotides in length, at least 500 nucleotides in length, or a combination thereof.

[0264] In some embodiments, identifying the sample origin of the at least one cell may include identifying the sample origin of the plurality of barcoded targets based on the sample-indexed sequence of at least one barcoded sample-indexed oligonucleotide. Barcoding the sample-indexed oligonucleotides with the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides may include probabilistically barcoding the sample-indexed oligonucleotides with the plurality of probabilistic barcodes to generate the plurality of probabilistic barcoded sample-indexed oligonucleotides.

[0265] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, each of the plurality of barcodes comprising a cell label, and at least two of the plurality of barcodes comprising an identical cell label sequence; and acquiring sequencing data for the barcoded targets. Barcoding a plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. Prior to acquiring sequencing data for the plurality of barcoded targets, the method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding a plurality of cellular targets using a plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding a plurality of cellular targets using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0266] In some embodiments, each of the plurality of sample indexing compositions comprises a cellular component binding reagent. The sample indexing sequences of sample indexing oligonucleotides associated with two or more cellular component binding reagents may be identical. The sample indexing sequences of sample indexing oligonucleotides associated with two or more cellular component binding reagents may comprise different sequences. Each of the plurality of sample indexing compositions may comprise two or more cellular component binding reagents. In some embodiments, the cellular component is an antigen. The cellular component binding reagent may be an antibody.

[0267] Disclosed herein is a method for sample identification. In some embodiments, the method includes the steps of contacting one or more cells from each of a plurality of samples with one sample-indexing composition from a plurality of sample-indexing compositions, wherein each of the one or more cells comprises one or more binding targets, each of the plurality of sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more binding targets, the sample-indexing oligonucleotide comprises a sample-indexing sequence, and the sample-indexing sequences of at least two of the plurality of sample-indexing compositions comprise different sequences; barcoding the sample-indexing oligonucleotides using a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides; obtaining sequencing data of the plurality of barcoded sample-indexing oligonucleotides using a plurality of daisy-chained amplification primers; and identifying the sample origin of at least one of the one or more cells based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides. The method may include removing unbound sample indexing compositions of the plurality of sample indexing compositions.

[0268] In some embodiments, the sample indexing sequence is at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, or at least 128 nucleotides in length, or a combination thereof. The sample indexing oligonucleotide may be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 200-500 nucleotides in length, about 500 nucleotides in length, or a combination thereof. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0269] In some embodiments, the cellular component binding reagent comprises a cell surface binding reagent, an antibody, a tetramer, an aptamer, a protein scaffold, an integrin, or a combination thereof. The sample-indexing oligonucleotide may be attached to the cellular component binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the cellular component binding reagent. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0270] In some embodiments, at least one sample of the plurality of samples comprises a single cell. At least one of the one or more cellular component targets may be expressed on a cell surface. One sample of the plurality of samples may comprise a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0271] In some embodiments, removing unbound sample-indexing composition comprises washing one or more cells from each of the plurality of samples with a wash buffer. The method may comprise lysing one or more cells from each of the plurality of samples. The sample-indexing oligonucleotide may be configured to be detachable or non-detachable from the cellular component binding reagent. The method may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent. The detaching the sample-indexing oligonucleotide may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0272] In some embodiments, the sample indexing oligonucleotide is not homologous to the genomic sequence of cells of the plurality of samples. The sample indexing oligonucleotide may comprise a molecular label sequence, a poly(A) region, or a combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(A) tail. The sample indexing oligonucleotide may comprise a molecular label.

[0273] In some embodiments, the cellular component target is or includes a carbohydrate, lipid, protein, extracellular protein, cell surface protein, cell marker, B cell receptor, T cell receptor, major histocompatibility complex, tumor antigen, receptor, integrin, intracellular protein, or any combination thereof. The cellular component target can be selected from a group including 10 to 100 different cellular component targets. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having the same sequence. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having different sample indexing sequences. The sample indexing composition of the multiple sample indexing compositions can include a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent can be the same.

[0274] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence. The molecular beacon sequences of at least two barcodes of the plurality of barcodes may comprise different molecular beacon sequences. The barcode may comprise a cell beacon, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0275] In some embodiments, the plurality of barcodes are encapsulated in particles. The particles may be beads. At least one barcode of the plurality of barcodes may be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof. The particles may be degradable. The beads may be selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise at least 10,000 barcodes. In some embodiments, the barcodes of the particles may comprise molecular beacon sequences selected from at least 1,000 or 10,000 different molecular beacon sequences. The molecular beacon sequences of the barcodes may comprise random sequences.

[0276] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes includes contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0277] In some embodiments, the method includes amplifying a plurality of barcoded sample-indexing oligonucleotides to generate a plurality of amplicons. Amplifying a plurality of barcoded sample-indexing oligonucleotides may include amplifying at least a portion of a molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR). Amplifying a plurality of barcoded sample-indexing oligonucleotides may include amplifying the plurality of barcoded sample-indexing oligonucleotides using a plurality of daisy-chained amplification primers to generate a plurality of daisy-chained extension amplicons. Acquiring sequencing data for the plurality of barcoded sample-indexing oligonucleotides may include acquiring sequencing data for the plurality of daisy-chained extension amplicons. Acquiring sequencing data may include sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

[0278] In some embodiments, a daisy-chained amplification primer of a plurality of daisy-chained amplification primers comprises a barcoded sample index-added oligonucleotide binding region and an overhang region, and the barcoded sample index-added oligonucleotide binding region is capable of binding to the daisy-chained sample index-added region of the sample index-added oligonucleotide. The barcoded sample index-added oligonucleotide binding region may be at least 20 nucleotides in length, at least 30 nucleotides in length, about 40 nucleotides in length, at least 40 nucleotides in length, about 50 nucleotides in length, or a combination thereof. Two daisy-chained amplification primers of a plurality of daisy-chained amplification primers may comprise barcoded sample index-added oligonucleotide binding regions having the same sequence. The plurality of daisy-chained amplification primers may comprise barcoded sample index-added oligonucleotide binding regions having the same sequence. The overhang region may be at least 50 nucleotides in length, at least 100 nucleotides in length, at least 150 nucleotides in length, about 150 nucleotides in length, at least 200 nucleotides in length, or a combination thereof. The overhang region may comprise a daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having the same daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having two daisy-chained amplification primer barcode sequences. The overhang regions of the plurality of daisy-chained amplification primers may comprise different daisy-chained amplification primer barcode sequences.The daisy-chained extension amplicons of the plurality of daisy-chained extension amplicons can be at least 250 nucleotides in length, at least 300 nucleotides in length, at least 350 nucleotides in length, at least 400 nucleotides in length, about 400 nucleotides in length, at least 450 nucleotides in length, at least 500 nucleotides in length, or a combination thereof.

[0279] In some embodiments, identifying the sample origin of the at least one cell may include identifying the sample origin of the plurality of barcoded targets based on the sample-indexed sequence of at least one barcoded sample-indexed oligonucleotide. Barcoding the sample-indexed oligonucleotides with the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides may include probabilistically barcoding the sample-indexed oligonucleotides with the plurality of probabilistic barcodes to generate the plurality of probabilistic barcoded sample-indexed oligonucleotides.

[0280] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, each of the plurality of barcodes comprising a cell label, and at least two of the plurality of barcodes comprising an identical cell label sequence; and acquiring sequencing data for the barcoded targets. Barcoding a plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target binding regions of the barcodes; and reverse transcribing the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. The method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to acquiring sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include amplifying the barcoded targets by polymerase chain reaction (PCR). Barcoding a plurality of cellular targets using a plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding a plurality of cellular targets using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

[0281] In some embodiments, each of the plurality of sample-indexing compositions comprises a cellular component binding reagent. The sample-indexing sequences of the sample-indexing oligonucleotides associated with two or more cellular component binding reagents may be identical. The sample-indexing sequences of the sample-indexing oligonucleotides associated with two or more cellular component binding reagents may comprise different sequences. Each of the plurality of sample-indexing compositions may comprise two or more cellular component binding reagents.

[0282] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with a sample indexing composition from a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more cellular component targets, each of the plurality of sample indexing compositions comprises a cellular component binding reagent associated with a sample indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets, the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; and identifying the sample origin of at least one of the one or more cells based on the sample indexing sequence of at least one sample indexing oligonucleotide of the plurality of sample indexing compositions and a plurality of daisy-chained amplification primers. Identifying the sample origin of at least one cell may include: barcoding sample-indexing oligonucleotides of a plurality of sample-indexing compositions with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides; obtaining sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides in the sequencing data. The method may, for example, include removing unbound sample-indexing compositions of the plurality of sample-indexing compositions.

[0283] In some embodiments, the sample indexing sequences are at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, or at least 128 nucleotides in length, or a combination thereof. The sample indexing oligonucleotides can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 200-500 nucleotides in length, about 500 nucleotides in length, or a combination thereof. The sample indexing sequences of at least 10 sample indexing compositions of the plurality of sample indexing compositions can comprise different sequences. The sample indexing sequences of at least 100 or 1000 sample indexing compositions of the plurality of sample indexing compositions can comprise different sequences.

[0284] In some embodiments, the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample-indexing oligonucleotide may be attached to the cellular component binding reagent via a linker. The oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the cellular component binding reagent. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0285] In some embodiments, at least one sample of the plurality of samples comprises a single cell. At least one of the one or more cellular component targets may be expressed on a cell surface. One sample of the plurality of samples may comprise a plurality of cells, tissues, tumor samples, or any combination thereof. The plurality of samples may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0286] In some embodiments, removing unbound sample-indexing composition comprises washing one or more cells from each of the plurality of samples with a wash buffer. The method may comprise lysing one or more cells from each of the plurality of samples. The sample-indexing oligonucleotide may be configured to be detachable or non-detachable from the cellular component binding reagent. The method may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent. The detaching the sample-indexing oligonucleotide may comprise detaching the sample-indexing oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment (e.g., using a reducing agent such as dithiothreitol), heating, enzymatic treatment, or any combination thereof.

[0287] In some embodiments, the sample indexing oligonucleotide is not homologous to the genomic sequence of cells of the plurality of samples. The sample indexing oligonucleotide may comprise a molecular label sequence, a poly(A) region, or a combination thereof. The sample indexing oligonucleotide may comprise a sequence complementary to the capture sequence of at least one barcode of the plurality of barcodes. The target binding region of the barcode may comprise a capture sequence. The target binding region may comprise a poly(dT) region. The sequence of the sample indexing oligonucleotide complementary to the capture sequence of the barcode may comprise a poly(A) tail. The sample indexing oligonucleotide may comprise a molecular label.

[0288] In some embodiments, the cellular component target is or includes a carbohydrate, lipid, protein, extracellular protein, cell surface protein, cell marker, B cell receptor, T cell receptor, major histocompatibility complex, tumor antigen, receptor, intracellular protein, or any combination thereof. The cellular component target can be selected from a group including 10 to 100 different cellular component targets. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having the same sequence. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having different sample indexing sequences. The sample indexing composition of the multiple sample indexing compositions can include a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent can be the same.

[0289] In some embodiments, identifying the sample origin of the at least one cell includes barcoding sample-indexing oligonucleotides of a plurality of sample-indexing compositions with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides; obtaining sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides.

[0290] In some embodiments, a barcode of the plurality of barcodes comprises a target binding region and a molecular beacon sequence. The molecular beacon sequences of at least two barcodes of the plurality of barcodes may comprise different molecular beacon sequences. The barcode may comprise a cell beacon, a binding site for a universal primer, or any combination thereof. The target binding region may comprise a poly(dT) region.

[0291] In some embodiments, the plurality of barcodes are immobilized on particles. At least one barcode of the plurality of barcodes can be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof. The particles can be degradable. The particles can be beads. The beads can be selected from the group consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and any combination thereof. The particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The particles may comprise at least 10,000 barcodes. In some embodiments, the barcodes of the particles may comprise molecular beacon sequences selected from at least 1,000 or 10,000 different molecular beacon sequences. The molecular beacon sequences of the barcodes may comprise random sequences.

[0292] In some embodiments, barcoding the sample-index-added oligonucleotides with a plurality of barcodes may include contacting the plurality of barcodes with the sample-index-added oligonucleotides to generate barcodes hybridized to the sample-index-added oligonucleotides; and extending the barcodes hybridized to the sample-index-added oligonucleotides to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a DNA polymerase to generate a plurality of barcoded sample-index-added oligonucleotides. Extending the barcodes may include extending the barcodes with a reverse transcriptase to generate a plurality of barcoded sample-index-added oligonucleotides.

[0293] In some embodiments, amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying the plurality of barcoded sample-indexing oligonucleotides using polymerase chain reaction (PCR). Amplifying the plurality of barcoded sample-indexing oligonucleotides may comprise amplifying at least a portion of a molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides. Amplifying the plurality of barcoded sample-indexing oligonucleotides may comprise amplifying the plurality of barcoded sample-indexing oligonucleotides using a plurality of daisy-chained amplification primers to generate a plurality of daisy-chained extension amplicons. Acquiring sequencing data of the plurality of barcoded sample-indexing oligonucleotides may comprise acquiring sequencing data of the plurality of daisy-chained extension amplicons. Acquiring sequencing data may comprise sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

[0294] In some embodiments, a daisy-chained amplification primer of a plurality of daisy-chained amplification primers comprises a barcoded sample index-added oligonucleotide binding region and an overhang region, and the barcoded sample index-added oligonucleotide binding region is capable of binding to the daisy-chained sample index-added region of the sample index-added oligonucleotide. The barcoded sample index-added oligonucleotide binding region may be at least 20 nucleotides in length, at least 30 nucleotides in length, about 40 nucleotides in length, at least 40 nucleotides in length, about 50 nucleotides in length, or a combination thereof. Two daisy-chained amplification primers of a plurality of daisy-chained amplification primers may comprise barcoded sample index-added oligonucleotide binding regions having the same sequence. The plurality of daisy-chained amplification primers may comprise barcoded sample index-added oligonucleotide binding regions having the same sequence. The overhang region may be at least 50 nucleotides in length, at least 100 nucleotides in length, at least 150 nucleotides in length, about 150 nucleotides in length, at least 200 nucleotides in length, or a combination thereof. The overhang region may comprise a daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having the same daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having two daisy-chained amplification primer barcode sequences. The overhang regions of the plurality of daisy-chained amplification primers may comprise different daisy-chained amplification primer barcode sequences.A daisy-chained extension amplicon of the plurality of daisy-chained extension amplicons can be at least 250 nucleotides in length, at least 300 nucleotides in length, at least 350 nucleotides in length, at least 400 nucleotides in length, at least 450 nucleotides in length, at least 500 nucleotides in length, or a combination thereof. A daisy-chained extension amplicon of the plurality of daisy-chained extension amplicons can be about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 600 nucleotides in length, or a range between any two of these values.

[0295] In some embodiments, barcoding sample-indexing oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexing oligonucleotides comprises probabilistic barcoding sample-indexing oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded sample-indexing oligonucleotides. Identifying the sample origin of the at least one cell may comprise identifying the presence or absence of a sample-indexing sequence of at least one sample-indexing oligonucleotide of the plurality of sample-indexing compositions. Identifying the presence or absence of a sample-indexing sequence may comprise replicating at least one sample-indexing oligonucleotide to generate a plurality of replicated sample-indexing oligonucleotides; obtaining sequencing data of the plurality of replicated sample-indexing oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of the replicated sample-indexing oligonucleotide of the plurality of sample-indexing oligonucleotides that corresponds to the at least one barcoded sample-indexing oligonucleotide in the sequencing data.

[0296] In some embodiments, replicating at least one sample-indexed oligonucleotide to generate a plurality of replicated sample-indexed oligonucleotides comprises ligating a replication adapter to the at least one barcoded sample-indexed oligonucleotide prior to replicating the at least one barcoded sample-indexed oligonucleotide. Replicating at least one barcoded sample-indexed oligonucleotide may comprise replicating the at least one barcoded sample-indexed oligonucleotide using a replication adapter ligated to the at least one barcoded sample-indexed oligonucleotide to generate a plurality of replicated sample-indexed oligonucleotides.

[0297] In some embodiments, replicating at least one sample-indexing oligonucleotide to generate a plurality of replicated sample-indexing oligonucleotides comprises: contacting a capture probe with the at least one sample-indexing oligonucleotide to generate a capture probe hybridized to the sample-indexing oligonucleotide prior to replicating the at least one barcoded sample-indexing oligonucleotide; and extending the capture probe hybridized to the sample-indexing oligonucleotide to generate a sample-indexing oligonucleotide associated with the capture probe. Replicating at least one sample-indexing oligonucleotide may comprise replicating the sample-indexing oligonucleotide associated with the capture probe to generate a plurality of replicated sample-indexing oligonucleotides.

[0298] In some embodiments, the method includes barcoding a plurality of cellular targets with a plurality of barcodes to generate a plurality of barcoded targets, each of the plurality of barcodes comprising a cell label, and at least two barcodes of the plurality of barcodes comprising an identical cell label sequence; and acquiring sequencing data for the barcoded targets. Identifying the sample origin of at least one barcoded sample-indexing oligonucleotide may include identifying the sample origin of the plurality of barcoded targets based on the sample-indexing sequence of the at least one barcoded sample-indexing oligonucleotide. Barcoding the plurality of targets with a plurality of barcodes to generate a plurality of barcoded targets may include contacting copies of the targets with target-binding regions of the barcodes; and reverse-transcribe the plurality of targets with the plurality of barcodes to generate a plurality of reverse-transcribed targets. The method may include amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to acquiring sequencing data for the plurality of barcoded targets. Amplifying the barcoded target to generate a plurality of amplified barcoded targets may include amplifying the barcoded target by polymerase chain reaction (PCR). Barcoding the plurality of cellular targets with the plurality of barcodes to generate a plurality of barcoded targets may include probabilistically barcoding the plurality of cellular targets with the plurality of stochastic barcodes to generate a plurality of stochastically barcoded targets.

[0299] In some embodiments, each of the plurality of sample-indexing compositions comprises a cellular component binding reagent. The sample-indexing sequences of the sample-indexing oligonucleotides associated with two or more cellular component binding reagents may be identical. The sample-indexing sequences of the sample-indexing oligonucleotides associated with two or more cellular component binding reagents may comprise different sequences. Each of the plurality of sample-indexing compositions may comprise two or more cellular component binding reagents.

[0300] Disclosed herein is a kit comprising a plurality of sample indexing compositions; and a plurality of daisy-chained amplification primers. Each of the plurality of sample indexing compositions may comprise two or more cellular component binding reagents. Each of the two or more cellular component binding reagents may be associated with a sample indexing oligonucleotide. At least one of the two or more cellular component binding reagents may be capable of specifically binding to at least one cellular component target. The sample indexing oligonucleotide may comprise a sample indexing sequence for identifying the sample origin of one or more cells of the sample. The sample indexing oligonucleotide may comprise a daisy-chained amplification primer binding sequence. The sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences.

[0301] The daisy-chained amplification primers of the plurality of daisy-chained amplification primers can comprise a barcoded sample indexing oligonucleotide binding region and an overhang region, and the barcoded sample indexing oligonucleotide binding region can bind to the daisy-chained sample indexing region of the sample indexing oligonucleotide. The barcoded sample indexing oligonucleotide binding region can be at least 20 nucleotides in length, at least 30 nucleotides in length, about 40 nucleotides in length, at least 40 nucleotides in length, about 50 nucleotides in length, or a combination thereof. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers can comprise barcoded sample indexing oligonucleotide binding regions with the same sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers can comprise barcoded sample indexing oligonucleotide binding regions with different sequences. The barcoded sample indexing oligonucleotide binding region can comprise a daisy-chained amplification primer binding sequence, its complement, its reverse complement, or a combination thereof. The daisy-chained amplification primer binding sequences of at least two sample indexing compositions of the plurality of sample indexing compositions can comprise the same sequence. The daisy-chained amplification primer binding sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. The overhang region may be at least 50 nucleotides in length, at least 100 nucleotides in length, at least 150 nucleotides in length, about 150 nucleotides in length, or a combination thereof. The overhang region may be at least 200 nucleotides in length. The overhang region may comprise a daisy-chained amplification primer barcode sequence. Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers may comprise overhang regions having the same daisy-chained amplification primer barcode sequence.Two daisy-chained amplification primers of the plurality of daisy-chained amplification primers can comprise overhang regions having two daisy-chained amplification primer barcode sequences. The overhang regions of the plurality of daisy-chained amplification primers can comprise different daisy-chained amplification primer barcode sequences.

[0302] In some embodiments, the sample indexing sequence is at least 6 nucleotides in length, 25-45 nucleotides in length, about 128 nucleotides in length, or at least 128 nucleotides in length, or a combination thereof. The sample indexing oligonucleotide can be about 50 nucleotides in length, about 100 nucleotides in length, about 200 nucleotides in length, at least 200 nucleotides in length, less than about 200-300 nucleotides in length, about 200-500 nucleotides in length, about 500 nucleotides in length, or a combination thereof. The sample indexing sequences of at least 10, 100, or 1000 sample indexing compositions of the plurality of sample indexing compositions comprise different sequences.

[0303] In some embodiments, the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof. The sample indexing oligonucleotide may be attached to the cellular component binding reagent via a linker. At least one sample indexing oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly attached to the cellular component binding reagent molecule. The chemical group may be selected from the group consisting of a UV photocleavable group, a disulfide bond, streptavidin, biotin, an amine, and any combination thereof.

[0304] In some embodiments, the sample indexing oligonucleotide is not homologous to a genomic sequence of a species. The sample indexing oligonucleotide may comprise a molecular beacon sequence, a poly(A) tract, or a combination thereof. In some embodiments, at least one sample of the plurality of samples may comprise a single cell, multiple cells, a tissue, a tumor sample, or any combination thereof. The sample may comprise a mammalian sample, a bacterial sample, a viral sample, a yeast sample, a fungal sample, or any combination thereof.

[0305] In some embodiments, the cellular component target is or includes a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, or any combination thereof. The cellular component target can be selected from a group comprising 10 to 100 different cellular component targets. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having the same sequence. The cellular component binding reagent can be associated with two or more sample indexing oligonucleotides having different sample indexing sequences. The sample indexing composition of the plurality of sample indexing compositions can include a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide. The cellular component binding reagent and the second cellular component binding reagent can be the same. [Brief explanation of the drawings]

[0306] [Figure 1] 1 illustrates a non-limiting exemplary probabilistic barcode. [Figure 2] 1 illustrates a non-limiting exemplary workflow for probabilistic barcoding and digital counting. [Figure 3] FIG. 1 is a schematic diagram showing a non-limiting exemplary process for creating an indexed library of probabilistic barcoded targets from multiple targets. [Figure 4]1 shows a schematic diagram of an exemplary protein binding reagent (an antibody as shown herein) conjugated to an oligonucleotide comprising a unique identifier for the protein binding reagent. [Figure 5] FIG. 1 shows a schematic diagram of an exemplary binding reagent (antibody as shown herein) conjugated to an oligonucleotide containing a unique identifier for sample indexing to determine cells from the same or different samples. [Figure 6] FIG. 1 shows a schematic diagram of an exemplary workflow using oligonucleotide-conjugated antibodies for simultaneous measurement of protein and gene expression in a high-throughput manner. [Figure 7] FIG. 1 shows a schematic diagram of an exemplary workflow using oligonucleotide-conjugated antibodies for sample indexing. [Figure 8A1] FIG. 1 shows a schematic diagram of an exemplary workflow for daisy-chaining barcoded oligonucleotides. [Figure 8A2] FIG. 1 shows a schematic diagram of an exemplary workflow for daisy-chaining barcoded oligonucleotides. [Figure 8B1] FIG. 1 shows a schematic diagram of another exemplary workflow for daisy-chaining barcoded oligonucleotides. [Figure 8B2] FIG. 1 shows a schematic diagram of another exemplary workflow for daisy-chaining barcoded oligonucleotides. [Figure 9A] 1 shows a non-limiting exemplary schematic diagram of a particle functionalized with an oligonucleotide. [Figure 9B] 1 shows a non-limiting exemplary schematic diagram of a particle functionalized with an oligonucleotide. [Figure 9C] 1 shows a non-limiting exemplary schematic diagram of a particle functionalized with an oligonucleotide. [Figure 9D] 1 shows a non-limiting exemplary schematic diagram of a particle functionalized with an oligonucleotide. [Figure 9E] 1 shows a non-limiting exemplary schematic diagram of a particle functionalized with an oligonucleotide. [Figure 10A]FIG. 1 is a schematic diagram of an exemplary workflow for the use of oligonucleotide-functionalized particles for single-cell sequencing control. [Figure 10B] FIG. 1 is a schematic diagram of another exemplary workflow for the use of oligonucleotide-functionalized particles for single-cell sequencing control. [Figure 11] FIG. 1 shows a schematic of an exemplary workflow for the use of control oligonucleotide-conjugated antibodies to determine single-cell sequencing efficiency. [Figure 12] FIG. 1 shows another schematic diagram of an exemplary workflow for the use of control oligonucleotide-conjugated antibodies to determine single-cell sequencing efficiency. [Figures 13A-13B] 1 is a plot showing that control oligonucleotides can be used for cell counting. [Figure 13C] 1 is a plot showing that control oligonucleotides can be used for cell counting. [Figure 14A] 1 shows a schematic diagram of an exemplary workflow for determining interactions between cellular components (eg, proteins) using pairs of interaction determination compositions. [Figure 14B] 1 shows a schematic diagram of an exemplary workflow for determining interactions between cellular components (eg, proteins) using pairs of interaction determination compositions. [Figure 14C] 1 shows a schematic diagram of an exemplary workflow for determining interactions between cellular components (eg, proteins) using pairs of interaction determination compositions. [Figure 14D] 1 shows a schematic diagram of an exemplary workflow for determining interactions between cellular components (eg, proteins) using pairs of interaction determination compositions. [Figure 14E] 1 shows a schematic diagram of an exemplary workflow for determining interactions between cellular components (eg, proteins) using pairs of interaction determination compositions. [Figure 14F] 1 shows a schematic diagram of an exemplary workflow for determining interactions between cellular components (eg, proteins) using pairs of interaction determination compositions. [Figures 15A-15B]1 shows non-limiting exemplary designs of oligonucleotides for simultaneously determining protein and gene expression and for sample indexing. [Figures 15C-15D] 1 shows non-limiting exemplary designs of oligonucleotides for simultaneously determining protein and gene expression and for sample indexing. [Figure 16] FIG. 1 shows a schematic diagram of non-limiting exemplary oligonucleotide sequences for simultaneously determining protein and gene expression and for sample indexing. [Figures 17A-17B] 1 is a non-limiting exemplary tSNE projection plot showing the results of using oligonucleotide-conjugated antibodies to simultaneously measure CD4 protein expression and gene expression in a high-throughput manner. [Figures 17C-17D] 1 is a non-limiting exemplary tSNE projection plot showing the results of using oligonucleotide-conjugated antibodies to simultaneously measure CD4 protein expression and gene expression in a high-throughput manner. [Figures 17E-17F] 1 is a non-limiting exemplary tSNE projection plot showing the results of using oligonucleotide-conjugated antibodies to simultaneously measure CD4 protein expression and gene expression in a high-throughput manner. [Figures 18A-18F] 1 is a non-limiting exemplary bar graph showing CD4 mRNA and protein expression in CD4 T cells, CD8 T cells, and myeloid cells. [Figure 19] 1 is a non-limiting exemplary bar graph showing increased detection sensitivity for CD4 protein levels at higher antibody:oligonucleotide ratios at similar sequencing depth, with a 1:3 ratio performing better than 1:1 and 1:2 ratios. [Figures 20A-20D] 1 is a plot showing CD4 protein expression on the cell surface of cells sorted using flow cytometry. [Figures 21A-21F] 1 is a non-limiting exemplary bar graph showing CD4 mRNA and protein expression in CD4 T cells, CD8 T cells, and bone marrow cells of two samples. [Figure 22] 1 is a non-limiting exemplary bar graph showing the detection sensitivity for CD4 protein levels determined using different sample preparation protocols at a 1:3 antibody:oligonucleotide ratio. [Figure 23] 1 shows a non-limiting exemplary experimental design for performing sample indexing and determining the effect of the length of the sample indexing oligonucleotide and the cleavability of the sample indexing oligonucleotide on sample indexing. [Figure 24A] 1 is a non-limiting exemplary tSNE plot showing that three types of anti-CD147 antibodies conjugated with various sample-indexed oligonucleotides (cleavable 95-mer, non-cleavable 95-mer, and cleavable 200-mer) can be used to determine the protein expression level of CD147. [Figure 24B] 1 is a non-limiting exemplary tSNE plot showing that three types of anti-CD147 antibodies conjugated with various sample-indexed oligonucleotides (cleavable 95-mer, non-cleavable 95-mer, and cleavable 200-mer) can be used to determine the protein expression level of CD147. [Figure 24C] 1 is a non-limiting exemplary tSNE plot showing that three types of anti-CD147 antibodies conjugated with various sample-indexed oligonucleotides (cleavable 95-mer, non-cleavable 95-mer, and cleavable 200-mer) can be used to determine the protein expression level of CD147. [Figure 25] 1 is a non-limiting exemplary tSNE plot with an overlay of GAPDH expression per cell. [Figure 26A] 1 is a non-limiting exemplary histogram showing the number of molecules of sample-indexed oligonucleotide detected using three types of sample-indexed oligonucleotide. [Figure 26B] 1 is a non-limiting exemplary histogram showing the number of molecules of sample-indexed oligonucleotide detected using three types of sample-indexed oligonucleotide. [Figure 26C] 1 is a non-limiting exemplary histogram showing the number of molecules of sample-indexed oligonucleotide detected using three types of sample-indexed oligonucleotide. [Figure 27A] 1 shows non-limiting exemplary plots and bar graphs showing that CD147 expression was higher in dividing cells. [Figure 27B] 1 shows non-limiting exemplary plots and bar graphs showing that CD147 expression was higher in dividing cells. [Figure 27C] 1 shows non-limiting exemplary plots and bar graphs showing that CD147 expression was higher in dividing cells. [Figure 28A] 1 is a non-limiting exemplary tSNE projection plot showing that sample indexing can be used to identify cells in various samples. [Figure 28B] 1 is a non-limiting exemplary tSNE projection plot showing that sample indexing can be used to identify cells in various samples. [Figure 28C] 1 is a non-limiting exemplary tSNE projection plot showing that sample indexing can be used to identify cells in various samples. [Figure 29A] 1 is a non-limiting exemplary histogram of sample-indexed sequences per cell based on the number of molecules of sample-indexed oligonucleotide determined. [Figure 29B] 1 is a non-limiting exemplary histogram of sample-indexed sequences per cell based on the number of molecules of sample-indexed oligonucleotide determined. [Figure 29C] 1 is a non-limiting exemplary histogram of sample-indexed sequences per cell based on the number of molecules of sample-indexed oligonucleotide determined. [Figure 30A]1 is a non-limiting exemplary plot comparing cell type annotation determined based on mRNA expression of CD3D (for Jurkat cells) and JCHAIN ​​(for Ramos cells) and sample indexing of Jurkat and Ramos cells. [Figure 30B] 1 is a non-limiting exemplary plot comparing cell type annotation determined based on mRNA expression of CD3D (for Jurkat cells) and JCHAIN ​​(for Ramos cells) and sample indexing of Jurkat and Ramos cells. [Figure 30C] 1 is a non-limiting exemplary plot comparing cell type annotation determined based on mRNA expression of CD3D (for Jurkat cells) and JCHAIN ​​(for Ramos cells) and sample indexing of Jurkat and Ramos cells. [Figure 30D] 1 is a non-limiting exemplary plot comparing cell type annotation determined based on mRNA expression of CD3D (for Jurkat cells) and JCHAIN ​​(for Ramos cells) and sample indexing of Jurkat and Ramos cells. [Figure 31A] 31A and 31B are non-limiting tSNE projection plots of the mRNA expression profiles of Jurkat and Ramos cells with overlays of mRNA expression of CD3D and JCHAIN ​​(FIG. 31A), JCHAIN ​​(FIG. 31B), and CD3D (FIG. 31C). [Figure 31B] 31A and 31B are non-limiting tSNE projection plots of the mRNA expression profiles of Jurkat and Ramos cells with overlays of mRNA expression of CD3D and JCHAIN ​​(FIG. 31A), JCHAIN ​​(FIG. 31B), and CD3D (FIG. 31C). [Figure 31C] 31A and 31B are non-limiting tSNE projection plots of the mRNA expression profiles of Jurkat and Ramos cells with overlays of mRNA expression of CD3D and JCHAIN ​​(FIG. 31A), JCHAIN ​​(FIG. 31B), and CD3D (FIG. 31C). [Figure 32]1 is a non-limiting exemplary tSNE projection plot of expression profiles of Jurkat and Ramos cells with an overlay of cell types determined using sample indexing with a DBEC cutoff of 250. [Figure 33A] 3A-3C are non-limiting exemplary bar graphs of the number of molecules of sample-indexed oligonucleotide per cell for Ramos and Jurkat cells ( FIG. 33 ), Ramos cells ( FIG. 33B ), and Jurkat cells ( FIG. 33C ) labeled with unlabeled or “short 3” sample-indexed oligonucleotide, “short 2” and “short 3” sample-indexed oligonucleotide, and “short 2” sample-indexed oligonucleotide. [Figure 33B] 3A-3C are non-limiting exemplary bar graphs of the number of molecules of sample-indexed oligonucleotide per cell for Ramos and Jurkat cells ( FIG. 33 ), Ramos cells ( FIG. 33B ), and Jurkat cells ( FIG. 33C ) labeled with unlabeled or “short 3” sample-indexed oligonucleotide, “short 2” and “short 3” sample-indexed oligonucleotide, and “short 2” sample-indexed oligonucleotide. [Figure 33C] 3A-3C are non-limiting exemplary bar graphs of the number of molecules of sample-indexed oligonucleotide per cell for Ramos and Jurkat cells ( FIG. 33 ), Ramos cells ( FIG. 33B ), and Jurkat cells ( FIG. 33C ) labeled with unlabeled or “short 3” sample-indexed oligonucleotide, “short 2” and “short 3” sample-indexed oligonucleotide, and “short 2” sample-indexed oligonucleotide. [Figure 34A] 1 is a non-limiting exemplary plot showing that less than 1% of single cells were labeled with both the "short 2" and "short 3" sample-indexed oligonucleotides. [Fig. 34B-34C]1 is a non-limiting exemplary plot showing that less than 1% of single cells were labeled with both the "short 2" and "short 3" sample-indexed oligonucleotides. [Figure 35A] 24A-24C are non-limiting exemplary tSNE plots showing batch effects on expression profiles of Jurkat and Ramos cells among samples prepared using various flow cells as outlined in FIG. 23. [Figure 35B] 24A-24C are non-limiting exemplary tSNE plots showing batch effects on expression profiles of Jurkat and Ramos cells among samples prepared using various flow cells as outlined in FIG. 23. [Figure 35C] 24A-24C are non-limiting exemplary tSNE plots showing batch effects on expression profiles of Jurkat and Ramos cells among samples prepared using various flow cells as outlined in FIG. 23. [Figures 36A1-36A3] 1 is a non-limiting exemplary plot showing determination of optimal dilution of antibody stock using dilution titration. [Figures 36A4-36A6] 1 is a non-limiting exemplary plot showing determination of optimal dilution of antibody stock using dilution titration. [Figures 36A7-36A9] 1 is a non-limiting exemplary plot showing determination of optimal dilution of antibody stock using dilution titration. [Figure 36B] 1 is a non-limiting exemplary plot showing determination of optimal dilution of antibody stock using dilution titration. [Figure 36C] 1 is a non-limiting exemplary plot showing determination of optimal dilution of antibody stock using dilution titration. [Figure 37] 1 shows a non-limiting exemplary experimental design for determining the staining concentration of an oligonucleotide-conjugated antibody such that the antibody oligonucleotide accounts for a desired percentage of total reads in sequencing data. [Figures 38A-38D] 1 is a non-limiting exemplary bioanalyzer trace showing peaks (indicated by arrows) consistent with the predicted size of antibody oligonucleotides. [Figure 39A1-39B1] 1 is a non-limiting exemplary histogram showing the number of molecules of antibody oligonucleotide detected for samples stained with different antibody dilutions and different percentages of antibody molecules conjugated with antibody oligonucleotide ("high temperature antibody"). [Figure 39A2-39B2] 1 is a non-limiting exemplary histogram showing the number of molecules of antibody oligonucleotide detected for samples stained with different antibody dilutions and different percentages of antibody molecules conjugated with antibody oligonucleotide ("high temperature antibody"). [Figure 39A3-39B3] 1 is a non-limiting exemplary histogram showing the number of molecules of antibody oligonucleotide detected for samples stained with different antibody dilutions and different percentages of antibody molecules conjugated with antibody oligonucleotide ("high temperature antibody"). [Figure 40A] Figure 40A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibody molecules can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 40A). Cells were stained with a mixture of 10% high-temperature antibody:90% low-temperature antibody prepared using a 1:100 diluted stock, resulting in a clear signal in the histogram (Figure 40B) showing the number of molecules of antibody oligonucleotide detected. Labeling of various cell types with antibody oligonucleotides is shown in Figure 40C. [Figure 40B] Figure 40A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibody molecules can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 40A). Cells were stained with a mixture of 10% high-temperature antibody:90% low-temperature antibody prepared using a 1:100 diluted stock, resulting in a clear signal in the histogram (Figure 40B) showing the number of molecules of antibody oligonucleotide detected. Labeling of various cell types with antibody oligonucleotides is shown in Figure 40C. [Figure 40C]Figure 40A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibody molecules can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 40A). Cells were stained with a mixture of 10% high-temperature antibody:90% low-temperature antibody prepared using a 1:100 diluted stock, resulting in a clear signal in the histogram (Figure 40B) showing the number of molecules of antibody oligonucleotide detected. Labeling of various cell types with antibody oligonucleotides is shown in Figure 40C. [Figure 41A] Figure 41A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibodies can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 41A). Staining cells with a mixture of 1% high-temperature antibody:99% low-temperature antibody prepared using a 1:100 diluted stock did not yield a clear signal in the histogram showing the number of molecules of antibody oligonucleotide detected (Figure 41B). Labeling of various cell types with antibody oligonucleotides is shown in Figure 41C. [Figure 41B] Figure 41A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibodies can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 41A). Staining cells with a mixture of 1% high-temperature antibody:99% low-temperature antibody prepared using a 1:100 diluted stock did not yield a clear signal in the histogram showing the number of molecules of antibody oligonucleotide detected (Figure 41B). Labeling of various cell types with antibody oligonucleotides is shown in Figure 41C. [Figure 41C]Figure 41A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibodies can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 41A). Staining cells with a mixture of 1% high-temperature antibody:99% low-temperature antibody prepared using a 1:100 diluted stock did not yield a clear signal in the histogram showing the number of molecules of antibody oligonucleotide detected (Figure 41B). Labeling of various cell types with antibody oligonucleotides is shown in Figure 41C. [Figure 42A] Figure 42A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibody molecules can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 42A). Cells were stained with a 1:800 diluted stock, resulting in a clear signal in the histogram (Figure 42B) showing the number of molecules of antibody oligonucleotide detected. Labeling of various cell types with antibody oligonucleotides is shown in Figure 42C. [Figure 42B] Figure 42A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibody molecules can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 42A). Cells were stained with a 1:800 diluted stock, resulting in a clear signal in the histogram (Figure 42B) showing the number of molecules of antibody oligonucleotide detected. Labeling of various cell types with antibody oligonucleotides is shown in Figure 42C. [Figure 42C]Figure 42A shows non-limiting exemplary plots demonstrating that oligonucleotide-conjugated anti-CD147 antibody molecules can be used to label various cell types. Cell types were determined using the expression profile of 488 genes in a blood panel (Figure 42A). Cells were stained with a 1:800 diluted stock, resulting in a clear signal in the histogram (Figure 42B) showing the number of molecules of antibody oligonucleotide detected. Labeling of various cell types with antibody oligonucleotides is shown in Figure 42C. [Figure 43A-43B] 11 is a plot showing the composition of control particle oligonucleotides in staining buffer and associated with detected control particles using the workflow shown in FIG. 10. [Figure 44A-44B] Bright field images of cells (Figure 44A, open circles) and control particles (Figure 44B, filled circles) in a hemocytometer. [Figure 45A-45B] Phase contrast (FIG. 45A, 10x) and fluorescence (FIG. 45B, 10x) images of control particles bound to oligonucleotide-conjugated antibodies associated with fluorophores. [Figure 46] 1 is an image showing cells and control particles loaded into the microwells of a cartridge. [Figure 47A] 1 is a plot showing that the use of antibody cocktails for sample indexing can increase labeling sensitivity. [Figure 47B] 1 is a plot showing that the use of antibody cocktails for sample indexing can increase labeling sensitivity. [Figure 47C] 1 is a plot showing that the use of antibody cocktails for sample indexing can increase labeling sensitivity. [Figure 48] 1 is a non-limiting exemplary plot showing that multiplets can be identified and removed from sequencing data using sample indexing. [Figure 49A]10A-B are non-limiting exemplary tSNE projection plots of CD45+ single cell expression profiles for 12 samples of 6 tissues from 2 mice identified as singlets or multiplets using sample-indexed oligonucleotides. [Figure 49B] Multiplets were identified using sample-indexed oligonucleotides and are shown removed in Figure 49A. Figure 49B is a non-limiting exemplary tSNE projection plot of CD45+ single cell expression profiles of 12 samples of 6 tissues from 2 mice. [Figure 50A] 10 is a non-limiting exemplary tSNE projection plot of the expression profile of CD45+ single cells from two mice in which multiplets identified using sample-indexed oligonucleotides were removed. [Fig. 50B-50C] 1 is a non-limiting exemplary pie chart showing that after removing multiplet expression profiles, two biological replicate mice showed similar expression profiles. [Figures 51A-51F] 1 is a non-limiting exemplary pie chart showing immune cell profiles of six different tissues with multiplet expression profiles in sequencing data identified and removed using sample-indexed oligonucleotides. [Figure 52A] 1 is a non-limiting exemplary graph showing expression profiles of macrophages, T cells, and B cells from six different tissues after multiplet expression profiles in the sequencing data have been identified and removed using sample-indexed oligonucleotides. [Figure 52B] 1 is a non-limiting exemplary graph showing expression profiles of macrophages, T cells, and B cells from six different tissues after multiplet expression profiles in the sequencing data have been identified and removed using sample-indexed oligonucleotides. [Figure 52C]1 is a non-limiting exemplary graph showing expression profiles of macrophages, T cells, and B cells from six different tissues after multiplet expression profiles in the sequencing data have been identified and removed using sample-indexed oligonucleotides. [Figure 53A] 1 is a non-limiting exemplary plot comparing macrophages in the colon and spleen. [Figure 53B] 1 is a non-limiting exemplary plot comparing macrophages in the colon and spleen. [Figure 53C] 1 is a non-limiting exemplary plot comparing macrophages in the colon and spleen. [Figure 53D] 1 is a non-limiting exemplary plot comparing macrophages in the colon and spleen. [Figure 54] 1 is a non-limiting exemplary plot showing that tagging cells with a sample indexing composition did not alter the mRNA expression profile in PBMCs. DETAILED DESCRIPTION OF THE INVENTION

[0307] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In these drawings, like numerals generally refer to like elements as identical unless the context requires otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It is to be understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and made a part of this disclosure.

[0308] All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety for the relevant art.

[0309] Quantification of small numbers of nucleic acids, such as messenger ribonucleic acid (mRNA) molecules, is clinically important, for example, to determine genes expressed at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can be extremely difficult, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR produces identical copies of molecules in each cycle. However, PCR can have problems, such as each molecule replicating with a stochastic probability, which varies depending on the PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Probabilistic barcodes with unique molecular labels (also called molecular beacons (MIs)) can be used to count the number of molecules and correct for amplification bias. Probabilistic barcoding, such as the Precise™ assay (Cellular Research, Inc., Palo Alto, CA), can correct for biases induced by the PCR and library generation steps by using molecular beacons (MLs) to label mRNA during reverse transcription (RT).

[0310] The Precise™ assay can use a non-depleted pool of stochastic barcodes bearing a large number of unique molecular labels (e.g., 6561-65536) on poly(T) oligonucleotides to hybridize with all poly(A)-mRNAs in a sample during the RT step. The stochastic barcodes may contain universal PCR priming sites. During RT, target gene molecules react randomly with the stochastic barcodes. Each target molecule may hybridize with the resulting stochastic barcode to generate a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules from the microwells of a microwell plate can be pooled into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to obtain the number of reads, the number of stochastic barcodes bearing unique molecular labels, and the number of mRNA molecules.

[0311] Methods for determining the mRNA expression profile of single cells can be performed in a massively parallel manner. For example, using the Precise™ assay, the mRNA expression profiles of more than 10,000 cells can be determined simultaneously. The number of single cells for analysis per sample (e.g., 100 or 1,000 single cells) can be less than the capabilities of current single-cell technologies. Pooling cells from different samples allows for improved utilization of the capabilities of current single-cell technologies, thus reducing the cost of wasted reagents and single-cell analysis. The present disclosure provides a sample indexing method for distinguishing cells from different samples for cDNA library generation for cell analysis, such as single-cell analysis. Pooling cells from different samples can minimize variability in cDNA library generation of cells from different samples, thus enabling more accurate comparison of different samples.

[0312] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with a sample indexing composition from a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more antigen targets, each of the plurality of sample indexing compositions comprises a protein binding reagent associated with a sample indexing oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the one or more antigen targets, the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; removing any unbound sample-indexing composition; barcoding (e.g., probabilistically barcoding) the sample-indexing oligonucleotides using a plurality of barcodes (e.g., probabilistic barcodes) to generate a plurality of barcoded sample-indexing oligonucleotides (e.g., probabilistic barcoded sample-indexing oligonucleotides); obtaining sequencing data of the plurality of barcoded sample-indexing oligonucleotides; and identifying a sample origin of at least one of the one or more cells based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides.

[0313] In some embodiments, the methods for sample identification disclosed herein include the steps of contacting one or more cells from each of a plurality of samples with one sample indexing composition of a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more cellular component targets, each of the plurality of sample indexing compositions comprises a cellular component binding reagent associated with a sample indexing oligonucleotide, the cellular component binding reagent being capable of specifically binding to at least one of the one or more cellular component targets, the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; The method includes removing unbound sample-index-added composition from the index-added composition; barcoding (e.g., probabilistic barcoding) sample-index-added oligonucleotides using a plurality of barcodes (e.g., probabilistic barcodes) to generate a plurality of barcoded sample-index-added oligonucleotides (e.g., probabilistic barcoded sample-index-added oligonucleotides); obtaining sequencing data of the plurality of barcoded sample-index-added oligonucleotides; and identifying the sample origin of at least one cell of the one or more cells based on the sample-index-added sequence of at least one barcoded sample-index-added oligonucleotide of the plurality of barcoded sample-index-added oligonucleotides.

[0314] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with one sample indexing composition from a plurality of sample indexing compositions, wherein each of the one or more cells comprises one or more antigen targets, each of the plurality of sample indexing compositions comprises a protein binding reagent associated with a sample indexing oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the one or more antigen targets, the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; removing unbound sample indexing compositions from the plurality of sample indexing compositions; and identifying the sample origin of at least one of the one or more cells based on the sample indexing sequence of at least one sample indexing oligonucleotide from the plurality of sample indexing compositions.

[0315] Disclosed herein are a plurality of sample indexing compositions. Each of the plurality of sample indexing compositions may comprise a protein binding reagent associated with a sample indexing oligonucleotide. The protein binding reagent may be capable of specifically binding to at least one antigen target. The sample indexing oligonucleotide may comprise a sample indexing sequence for identifying the sample origin of one or more cells of the sample. The sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences.

[0316] Disclosed herein is a method for sample identification. In some embodiments, the method includes contacting one or more cells from each of a plurality of samples with a sample indexing composition of a plurality of sample indexing compositions, wherein the one or more cells comprise one or more cellular component targets, each of the plurality of sample indexing compositions comprising a cellular component binding reagent (e.g., an antibody) associated with a sample indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets (e.g., a protein), the sample indexing oligonucleotide comprising a sample indexing sequence, and the sample indexing sequences of at least two of the plurality of sample indexing compositions comprise different sequences; and identifying the samples using the plurality of barcodes. The method includes barcoding pull-index oligonucleotides to generate a plurality of barcoded sample-indexed oligonucleotides; amplifying the plurality of barcoded sample-indexed oligonucleotides with a plurality of daisy-chained amplification primers to generate a plurality of daisy-chained extension amplicons; obtaining sequencing data of the plurality of daisy-chained extension amplicons, the sequencing data of the plurality of barcoded sample-indexed oligonucleotides; and identifying a sample origin of at least one cell of the one or more cells based on the sample-indexed sequence of at least one barcoded sample-indexed oligonucleotide of the plurality of barcoded sample-indexed oligonucleotides. The method may include removing unbound sample-indexed compositions from the plurality of sample-indexed compositions.

[0317] In some embodiments, the sample indexing methods, kits, and compositions disclosed herein can increase sample throughput (e.g., for rare samples with low cell counts, cells and heterogeneous cells are difficult to isolate), lower reagent costs, reduce technical errors and batch effects by performing library generation in a single-tube reaction, and / or identify multiple cells across samples during data analysis. In some embodiments, cells of tissues from various lymphoid and non-lymphoid organs can be tagged with different sample indexing compositions, for example, to increase sample throughput and reduce or minimize batch effects. In some embodiments, immune defense and tissue homeostasis and function can be investigated using the disclosed methods, kits, and compositions.

[0318] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0319] As used herein, the term "adapter" may refer to a sequence for facilitating the amplification or sequencing of a related nucleic acid. The related nucleic acid may include a target nucleic acid. The related nucleic acid may include one or more of a spatial label, a target label, a sample label, an index label, or a barcode sequence (e.g., a molecular label). The adapter may be linear. The adapter may be a pre-adenylated adapter. The adapter may be double-stranded or single-stranded. One or more adapters may be positioned at the 5' or 3' end of a nucleic acid. When an adapter includes a known sequence at the 5' and 3' ends, the known sequences may be the same or different sequences. An adapter located at the 5' and / or 3' end of a polynucleotide may be capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, the adapter includes a universal sequence. The universal sequence may be a region of nucleotide sequence common to two or more nucleic acid molecules. The two or more nucleic acid molecules may have regions of different sequences. Thus, for example, the 5' adapter may comprise an identical and / or universal nucleic acid sequence, and the 3' adapter may comprise an identical and / or universal sequence. A universal sequence that may be present in different members of a plurality of nucleic acid molecules may allow for the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair), or more universal sequences that may be present in different members of a collection of nucleic acid molecules may allow for the replication or amplification of multiple different sequences using at least one, two (e.g., a pair), or more single universal primers that are complementary to the universal sequence. Thus, a universal primer comprises a sequence that can hybridize to such a universal sequence. Target nucleic acid sequence-bearing molecules can be modified to attach universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of different target nucleic acid sequences. One or more universal primers bound to the target nucleic acids can provide sites for hybridization of the universal primers. The one or more universal primers bound to the target nucleic acids may be the same or different from each other.

[0320] As used herein, an antibody can be a full-length (e.g., naturally occurring or generated by conventional immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specific binding) portion of an immunoglobulin molecule, such as an antibody fragment.

[0321] In some embodiments, the antibody is a functional antibody fragment. For example, an antibody fragment can be a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, or sFv. An antibody fragment can bind to the same antigen recognized by the full-length antibody. Antibody fragments can include isolated fragments consisting of the variable regions of an antibody, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are connected by a peptide linker ("scFv protein"). Exemplary antibodies can include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (e.g., CD8, CD34, and CD45), and therapeutic antibodies.

[0322] As used herein, the terms "associated" or "associated with" can mean that two or more species are identifiable as co-located at a given time. Association can mean that two or more species are in similar containers. Association can be an informatic association. For example, digital information about two or more species is stored, and that information can be used to determine that one or more of these species are co-located. Association can also be a physical association. In some embodiments, two or more associated species are "tethered," "bound," or "immobilized" to each other or to a common solid or semi-solid surface. Association can refer to covalent or non-covalent means for attaching a label to a solid or semi-solid support, such as a bead. Association can be a covalent bond between a target and a label. Association can include hybridization between two molecules (such as a target molecule and a label).

[0323] As used herein, the term "complementary" can refer to the ability for precise pairing between two nucleotides. For example, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, the two nucleic acids are considered complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," where only some of the nucleotides bind, or complete, where complementarity exists across the entirety of the single-stranded molecules. If a first nucleotide sequence is complementary to a second nucleotide sequence, the first nucleotide sequence can be said to be the "complement" of the second sequence. If a first nucleotide sequence is complementary to the reverse of the second sequence (i.e., the order of the nucleotides is reversed), the first nucleotide sequence can be said to be the "reverse complement" of the second sequence. As used herein, the terms "complement," "complementary," and "reverse complement" can be used interchangeably. It is understood from this disclosure that when a molecule is capable of hybridizing to another molecule, it may be the complement of the hybridizing molecule.

[0324] As used herein, the term "digital counting" can refer to a method for estimating the number of target molecules in a sample. Digital counting can include determining the number of unique labels associated with targets in a sample. This method can be probabilistic in nature, transforming the problem of counting molecules from one of locating and identifying identical molecules to a series of digital present / absent problems involving the detection of a given set of labels.

[0325] As used herein, the term "label" can refer to a nucleic acid code associated with a target in a sample. A label can be, for example, a nucleic acid label. A label can be, in whole or in part, an amplifiable label. A label can be, in whole or in part, a sequenceable label. A label can be a portion of a naturally occurring nucleic acid that is individually identifiable. A label can be a known sequence. A label can include a nucleic acid sequence junction (e.g., a junction of a naturally occurring sequence and a non-naturally occurring sequence). As used herein, the term "label" can be used interchangeably with the terms "index," "tag," or "label tag." A label can convey information. For example, in various embodiments, a label can be used to determine sample identity, sample source, cell identity, and / or target.

[0326] As used herein, the term "non-depleted reservoir" can refer to a pool of barcodes (e.g., stochastic barcodes) composed of a wide variety of labels. A non-depleted reservoir can contain a large number of different barcodes so that when the non-depleted reservoir is associated with a pool of targets, each target is likely to be associated with a unique barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random selection and depends on the copy number of the same target molecule in the collection compared to the diversity of the labels. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and analysis of the number of detected barcodes then allows for the calculation of the number of target molecules present in the original collection or sample. If the ratio of the copy number of target molecules present to the number of unique barcodes is low, the labeled target molecules are highly unique (i.e., the probability that two or more target molecules are labeled with a given label is very low).

[0327] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or a fragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some examples of analogs include, but are not limited to, 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. The terms "nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" may be used interchangeably.

[0328] Nucleic acids can contain one or more modifications (e.g., base modifications, backbone modifications) to provide nucleic acids with new or improved characteristics (e.g., improved stability). Nucleic acids can contain nucleic acid affinity tags. Nucleosides can be base-sugar combinations. The base portion of a nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. In nucleosides that include a pentofuranosyl sugar, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups can covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound can then be further linked to form a circular compound. However, linear compounds are generally preferred. Additionally, linear compounds may have internal nucleotide base complementarity and therefore may fold to produce fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups can commonly be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone can be a 3' to 5' phosphodiester linkage.

[0329] Nucleic acids may contain modified backbones and / or modified internucleoside linkages. Modified backbones may include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates such as 3'-aminophosphoramidate and aminoalkylphosphoramidate, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates, typically having a 3'-5' linkage, a 2'-5' linkage analog, and those in which one or more internucleotide linkages have reverse polarity, such as a 3'→3', 5'→5', or 2'→2' linkage.

[0330] Nucleic acids can contain polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, which can include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0331] Nucleic acids may include nucleic acid mimetics. The term "mimetic" is intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups; replacement of only the furanose ring can be referred to as a sugar surrogate. Heterocyclic base moieties or modified heterocyclic base moieties can be retained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In PNA, the sugar backbone of a polynucleotide can be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleotides can be retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. The backbone in a PNA compound can contain two or more linked aminoethylglycine units, giving the PNA an amide-containing backbone. Heterocyclic base moieties can be directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone.

[0332] Nucleic acids can include morpholino backbone structures. For example, nucleic acids can include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages can replace the phosphodiester linkages.

[0333] Nucleic acids can contain linked morpholino units (e.g., morpholino nucleic acids) having heterocyclic bases attached to the morpholino ring. Linking groups can connect morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds can have fewer undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be linked using different linking groups. A further class of polynucleotide mimetics can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stabilities similar to those of native complexes. Further modifications can include locked nucleic acids (LNAs), in which a 2'-hydroxyl group is attached to the 4' carbon atom of the sugar ring to form a 2'-C,4'-C-oxymethylene linkage, forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2) group (where n is 1 or 2) bridging the 2' oxygen atom and the 4' carbon atom. LNAs and LNA analogs can exhibit very high duplex thermal stability with complementary nucleic acids (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility.

[0334] Nucleic acids may also include modifications or substitutions of nucleobases (often simply referred to as "bases"). As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C) and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, These may include cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoad...

Claims

1. 1. A method for sample identification, comprising: contacting one or more cells from each of a plurality of samples with one sample-indexing composition of a plurality of sample-indexing compositions; the one or more cells comprise one or more protein targets, and at least one sample indexing composition of the plurality of sample indexing compositions comprises two or more protein binding reagents, each of the two or more protein binding reagents being associated with a sample indexing oligonucleotide, and at least one of the two or more protein binding reagents being capable of specifically binding to at least one of the one or more protein targets; the sample indexing oligonucleotides comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; identifying a sample origin of at least one of the one or more cells based on the sample indexing sequence of at least one barcoded sample indexing oligonucleotide of the plurality of barcoded sample indexing oligonucleotides; A method comprising:

2. 1. A method for sample identification, comprising: contacting one or more cells from each of a plurality of samples with one sample-indexing composition of a plurality of sample-indexing compositions; each of the one or more cells comprises one or more cellular component targets; at least one sample indexing composition of the plurality of sample indexing compositions comprises two or more cellular component binding reagents, each of the two or more cellular component binding reagents being associated with a sample indexing oligonucleotide, and at least one of the two or more cellular component binding reagents being capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotides comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; identifying a sample origin of at least one of the one or more cells based on the sample indexing sequence of at least one barcoded sample indexing oligonucleotide of the plurality of barcoded sample indexing oligonucleotides; A method comprising:

3. 3. The method of claim 2, wherein the two or more cellular component binding reagents comprise two or more protein binding reagents, each of the two or more protein binding reagents being associated with the sample-indexing oligonucleotide associated with a corresponding cellular component binding reagent, and the one or more cellular component targets comprise one or more protein targets.

4. 4. The method of claim 1, wherein identifying the sample origin of the at least one cell comprises identifying the sample origin of the plurality of barcoded targets based on the sample indexing sequence of the at least one barcoded sample indexing oligonucleotide.

5. 1. A method for sample identification, comprising: contacting one or more cells from each of a plurality of samples with one sample-indexing composition of a plurality of sample-indexing compositions; each of the one or more cells comprises one or more cellular component targets; at least one sample indexing composition of the plurality of sample indexing compositions comprises two or more cellular component binding reagents, each of the two or more cellular component binding reagents being associated with a sample indexing oligonucleotide, and at least one of the two or more cellular component binding reagents being capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotides comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; and identifying the sample origin of at least one of the one or more cells based on the sample indexing sequence of at least one sample indexing oligonucleotide of the plurality of sample indexing compositions.

6. 6. The method of claim 5, wherein the two or more cellular component binding reagents comprise two or more protein binding reagents, each of the two or more protein binding reagents being associated with the sample-indexing oligonucleotide associated with a corresponding cellular component binding reagent, and the one or more cellular component targets comprise one or more protein targets.

7. identifying the sample origin of the at least one cell, barcoding the sample-indexed oligonucleotides of said plurality of sample-indexed compositions with the plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of at least one barcoded sample-indexing oligonucleotide of the plurality of barcoded sample-indexing oligonucleotides in the sequencing data.

8. 8. The method of claim 5, wherein identifying the sample origin of the at least one cell comprises identifying the presence or absence of the sample indexing sequence of at least one sample indexing oligonucleotide of the plurality of sample indexing compositions.

9. identifying the presence or absence of the sample indexing sequence, replicating said at least one sample-indexed oligonucleotide to generate a plurality of replicated sample-indexed oligonucleotides; obtaining sequencing data for the plurality of replicated sample-indexed oligonucleotides; and identifying the sample origin of the cell based on the sample-indexing sequence of a replicated sample-indexing oligonucleotide of the plurality of sample-indexing oligonucleotides that corresponds to the at least one barcoded sample-indexing oligonucleotide in the sequencing data.

10. replicating the at least one sample-indexing oligonucleotide to generate the plurality of replicated sample-indexing oligonucleotides comprises ligating a replication adapter to the at least one barcoded sample-indexing oligonucleotide before replicating the at least one barcoded sample-indexing oligonucleotide; 10. The method of claim 9, wherein replicating the at least one barcoded sample-indexing oligonucleotide comprises replicating the at least one barcoded sample-indexing oligonucleotide using the replication adapter ligated to the at least one barcoded sample-indexing oligonucleotide to generate the plurality of replicated sample-indexing oligonucleotides.

11. and replicating the at least one sample-indexing oligonucleotide to generate the plurality of replicated sample-indexing oligonucleotides, before replicating the at least one barcoded sample-indexing oligonucleotide. contacting a capture probe with said at least one sample-indexing oligonucleotide to produce a capture probe hybridized to said sample-indexing oligonucleotide; extending the capture probe hybridized to the sample-indexed oligonucleotide to generate a sample-indexed oligonucleotide associated with the capture probe; 10. The method of claim 9, wherein replicating the at least one sample-indexing oligonucleotide comprises replicating the sample-indexing oligonucleotide associated with the capture probe to generate the plurality of replicated sample-indexing oligonucleotides.

12. The method of any one of claims 1 to 11, wherein the sample index addition sequence is 6 to 60 nucleotides in length.

13. 13. The method of any one of claims 1 to 12, wherein the sample-indexing oligonucleotide is 50 to 500 nucleotides in length.

14. 14. The method of any one of claims 1 to 13, wherein the sample indexing sequences of at least 10, 100, 1000, or a combination thereof, of the plurality of sample indexing compositions comprise different sequences.

15. The method of any one of claims 1 to 14, wherein the protein-binding reagent or the cellular component-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof.

16. The method of any one of claims 1 to 15, wherein the sample-indexing oligonucleotide is attached to the cellular component binding reagent via a linker.

17. The method of any one of claims 1 to 16, wherein at least one sample of the plurality of samples comprises a single cell.

18. 18. The method of any one of claims 1 to 17, wherein at least one of the one or more protein targets or the one or more cellular component targets is on a cell surface.

19. 19. The method of any one of claims 1 to 18, comprising removing unbound sample indexing compositions of said plurality of sample indexing compositions.

20. 20. The method of claim 19, wherein removing the unbound sample-indexing composition comprises washing the one or more cells from each of the plurality of samples with a wash buffer.

21. 20. The method of claim 19, wherein removing the unbound sample indexing compositions comprises using flow cytometry to select cells bound to a cellular component binding reagent of at least one of the two sample indexing compositions.

22. The method of any one of claims 1 to 21, comprising lysing the one or more cells from each of the plurality of samples.

23. The method of any one of claims 1 to 22, wherein the sample-indexing oligonucleotide is configured to be detachable or non-detachable from the cellular component binding reagent.

24. The method of any one of claims 1 to 23, comprising the step of cleaving the sample-indexing oligonucleotide from the cellular component binding reagent.

25. 25. The method of claim 24, wherein cleaving the sample-indexing oligonucleotide comprises cleaving the sample-indexing oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof.

26. 26. The method of any one of claims 1 to 25, wherein the sample-indexing oligonucleotide is not homologous to any genomic sequence of the one or more cells, is homologous to a genomic sequence of a species, or a combination thereof.

27. 27. The method of claim 26, wherein the species is a non-mammalian species.

28. 28. The method of any one of claims 1 to 27, wherein one sample of the plurality of samples comprises a plurality of cells, tissues, tumor samples, or any combination thereof.

29. 29. The method of any one of claims 1 to 28, wherein the plurality of samples comprises mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof.

30. 30. The method of any one of claims 1 to 29, wherein the sample indexing oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the sequence of the sample indexing oligonucleotide.

31. 31. The method of claim 30, wherein the barcode comprises a target binding region that includes the capture sequence.

32. 32. The method of claim 31 , wherein the target binding region comprises a poly(dT) region.

33. 33. The method of any one of claims 30 to 32, wherein the sequence of the sample-indexing oligonucleotide complementary to the capture sequence comprises a poly(dA) region.

34. 34. The method of any one of claims 1 to 33, wherein the sample-indexing oligonucleotide comprises a molecular beacon sequence, a binding site for a universal primer, or both.

35. 35. The method of any one of claims 1 to 34, wherein the protein target or the cellular component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

36. 36. The method of any one of claims 1 to 35, wherein the protein target or the cellular component target is selected from a group comprising 10 to 100 different protein targets or cellular component targets.

37. 37. The method of any one of claims 1 to 36, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence.

38. 37. The method of any one of claims 1 to 36, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences.

39. The method of any one of claims 1 to 38, wherein the sample indexing composition of the plurality of sample indexing compositions comprises a second protein binding reagent that is not associated with the sample indexing oligonucleotide, or a second cellular component binding reagent that is not associated with the sample indexing oligonucleotide.

40. 40. The method of claim 39, wherein the protein-binding reagent and the second protein-binding reagent are the same, or the cellular component-binding reagent and the second cellular component-binding reagent are the same.

41. 41. The method of any one of claims 1 to 40, wherein each of the plurality of sample indexing compositions comprises the protein binding reagent or the cellular component binding reagent.

42. 41. The method of any one of claims 1 to 40, wherein the sample indexing sequences of the sample indexing oligonucleotides associated with the two or more protein binding reagents or the two or more cellular component binding reagents are identical.

43. 41. The method of any one of claims 1 to 40, wherein the sample indexing sequences of the sample indexing oligonucleotides associated with two or more protein-binding reagents or the two or more cellular component-binding reagents comprise different sequences.

44. 44. The method of any one of claims 1 to 43, wherein each of said plurality of sample indexing compositions comprises said two or more protein binding reagents or said two or more cellular component binding reagents.

45. a barcode of the plurality of barcodes comprising a target binding region and a molecular beacon sequence; 45. The method of any one of claims 1 to 44, wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences.

46. 46. ​​The method of claim 45, wherein the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

47. 47. The method of any one of claims 45 to 46, wherein the target binding region comprises a poly(dT) region.

48. The method of any one of claims 45 to 47, wherein the plurality of barcodes are associated with particles.

49. 49. The method of claim 48, wherein at least one barcode of the plurality of barcodes is immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or a combination thereof.

50. 50. The method of any one of claims 48 to 49, wherein the particles are disintegrating.

51. 51. The method of any one of claims 48 to 50, wherein the particles comprise beads.

52. 52. The method of any one of claims 48 to 51, wherein the particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof, or wherein the particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

53. 53. The method of any one of claims 48 to 52, wherein the particles comprise collapsible hydrogel beads.

54. 54. The method of any one of claims 48 to 53, wherein the barcode of the particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or a combination thereof, different molecular beacon sequences.

55. 55. The method of claim 54, wherein the molecular beacon sequence of the barcode comprises a random sequence.

56. 56. The method of any one of claims 48 to 55, wherein the particles comprise at least 10,000 barcodes.

57. barcoding said sample-indexed oligonucleotides using said plurality of barcodes, contacting the plurality of barcodes with the sample-indexed oligonucleotides to produce barcodes hybridized to the sample-indexed oligonucleotides; and extending the barcodes hybridized to the sample-indexed oligonucleotides to generate the plurality of barcoded sample-indexed oligonucleotides.

58. 58. The method of Claim 57, wherein extending the barcodes comprises extending the barcodes with a DNA polymerase to generate the plurality of barcoded sample-indexed oligonucleotides.

59. 58. The method of Claim 57, wherein extending the barcodes comprises extending the barcodes using a reverse transcriptase to generate the plurality of barcoded sample-indexed oligonucleotides.

60. 60. The method of any one of claims 57-59, comprising amplifying said plurality of barcoded, sample-indexed oligonucleotides to generate a plurality of amplicons.

61. 61. The method of Claim 60, wherein amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR).

62. 62. The method of any one of claims 60-61, wherein obtaining the sequencing data for the plurality of barcoded, sample-indexed oligonucleotides comprises obtaining sequencing data for the plurality of amplicons.

63. 63. The method of Claim 62, wherein obtaining the sequencing data comprises sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotide.

64. 64. The method of any one of claims 1 to 63, wherein barcoding the sample-indexed oligonucleotides with the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides with a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded sample-indexed oligonucleotides.

65. barcoding a plurality of targets of the cells using the plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell-labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell-labeling sequence; and obtaining sequencing data for said barcoded targets.

66. barcoding the plurality of targets using the plurality of barcodes to generate the plurality of barcoded targets; contacting a copy of the target with the target binding region of the barcode; and reverse transcribing said plurality of targets using said plurality of barcodes to generate a plurality of reverse transcribed targets.

67. 67. The method of any one of claims 65-66, comprising amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining the sequencing data for the plurality of barcoded targets.

68. 68. The method of claim 67, wherein amplifying the barcoded target to generate the plurality of amplified barcoded targets comprises amplifying the barcoded target by polymerase chain reaction (PCR).

69. 69. The method of any one of claims 65-68, wherein barcoding the plurality of targets of the cells using the plurality of barcodes to generate the plurality of barcoded targets comprises probabilistically barcoding the plurality of targets of the cells using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

70. A plurality of sample-indexed compositions, each of said plurality of sample indexing compositions comprises two or more cellular component binding reagents; each of the two or more cellular component binding reagents is associated with a sample-indexing oligonucleotide; at least one of the two or more cellular component binding reagents is capable of specifically binding to at least one cellular component target; the sample indexing oligonucleotide comprises a sample indexing sequence for identifying the sample origin of one or more cells of the sample; A plurality of sample indexing compositions, wherein the sample indexing sequences of at least two sample indexing compositions of said plurality of sample indexing compositions comprise different sequences.

71. 71. The plurality of sample indexing compositions of claim 70, wherein the sample indexing sequences are between 6 and 60 nucleotides in length.

72. 72. The plurality of sample indexing compositions of any one of claims 70-71, wherein the sample indexing oligonucleotides are 50-500 nucleotides in length.

73. 73. The plurality of sample indexing compositions of any one of claims 70 to 72, wherein the sample indexing sequences of at least 10, 100, 1000, or a combination thereof, of the plurality of sample indexing compositions comprise different sequences.

74. 74. The plurality of sample indexing compositions of any one of claims 70-73, wherein the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof.

75. 75. The plurality of sample indexing compositions of any one of claims 70 to 74, wherein the sample indexing oligonucleotides are attached to the cellular component binding reagents via a linker.

76. 76. The plurality of sample indexing compositions of any one of claims 70-75, wherein the sample indexing oligonucleotides are not homologous to any genomic sequence of the one or more cells.

77. 77. The multiple sample indexing composition of claim 76, wherein at least one sample of said multiple samples comprises a single cell, multiple cells, a tissue, a tumor sample, or any combination thereof.

78. 78. The plurality of sample indexing compositions of any one of claims 76-77, wherein the samples comprise mammalian samples, bacterial samples, viral samples, yeast samples, fungal samples, or any combination thereof.

79. 79. The plurality of sample indexing compositions of any one of claims 70-78, wherein the sample indexing oligonucleotide comprises a molecular beacon sequence, a poly(dA) region, or a combination thereof.

80. 80. The multiple sample indexing composition of any one of claims 70-79, wherein the cellular component target comprises a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, or any combination thereof.

81. 81. The multiple sample indexing composition of any one of claims 70-80, wherein the cellular component targets are selected from a group comprising 10 to 100 different cellular component targets.

82. 82. The plurality of sample-indexing compositions of any one of claims 70-81, wherein the cellular component binding reagents are associated with two or more sample-indexing oligonucleotides having the same sequence.

83. 82. The plurality of sample-indexing compositions of any one of claims 70 to 81, wherein the cellular component binding reagents are associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences.

84. A plurality of sample indexing compositions according to any one of claims 70 to 83, wherein the sample indexing composition of the plurality of sample indexing compositions comprises a second cellular component binding reagent that is not associated with the sample indexing oligonucleotide.

85. 85. The plurality of sample indexing compositions of claim 84, wherein the cellular component binding reagent and the second cellular component binding reagent are the same.

86. 1. A control particle composition comprising a plurality of control particle oligonucleotides associated with a control particle, A control particle composition, wherein each of the plurality of control particle oligonucleotides comprises a control barcode sequence and a poly(dA) region.

87. 87. The control particle composition of claim 86, wherein at least two of the plurality of control particle oligonucleotides comprise different control barcode sequences.

88. 88. The control particle composition of any one of claims 86 to 87, wherein the control barcode sequence is 6 to 60 nucleotides in length.

89. 89. The control particle composition of any one of claims 86 to 88, wherein the control particle oligonucleotide is 50 to 500 nucleotides in length. The control particle composition of any one of claims 86 to 89, wherein the control barcode sequences of at least 5, 10, 100, 1000, or a combination thereof, of the plurality of control particle oligonucleotides are identical.

90. 90. The control particle composition of any one of claims 86 to 89, wherein at least 3, 5, 10, 100, or a combination thereof, of the plurality of control particle oligonucleotides comprise different control barcode sequences.

91. the plurality of control particle oligonucleotides a plurality of first control particle oligonucleotides each comprising a first control barcode sequence; a plurality of second control particle oligonucleotides, each of which comprises a second control barcode sequence; 91. The control particle composition of any one of claims 86 to 90, wherein the first control barcode sequence and the second control barcode sequence have different sequences.

92. 92. The control particle composition of claim 91, wherein the number of said plurality of first control particle oligonucleotides and the number of said plurality of second control particle oligonucleotides are approximately the same.

93. 92. The control particle composition of claim 91, wherein the number of the first plurality of control particle oligonucleotides and the number of the second plurality of control particle oligonucleotides are different.

94. The control particle composition of claim 93, wherein the number of the first control particle oligonucleotides is at least 2 times, 10 times, 100 times, or a combination thereof greater than the number of the second control particle oligonucleotides.

95. 95. The control particle composition of any one of claims 86 to 94, wherein the control barcode sequence is not homologous to the genomic sequence of a species.

96. 95. The control particle composition of any one of claims 86 to 94, wherein the control barcode sequence is homologous to a genomic sequence of one species.

97. 97. The control particle composition of claim 96, wherein the species is a non-mammalian species.

98. 98. The control particle composition of any one of claims 86 to 97, wherein the control particle oligonucleotide comprises a molecular beacon sequence, a binding site for a universal primer, or both.

99. A control particle composition described in any one of claims 86 to 98, wherein at least one of the plurality of control particle oligonucleotides is associated with the control particle via a linker.

100. 100. The control particle composition of any one of claims 86 to 99, wherein the diameter of the control particle is about 1 to 1000 micrometers, about 10 to 100 micrometers, about 7.5 micrometers, or a combination thereof.

101. A control particle composition described in any one of claims 86 to 100, wherein the plurality of control particle oligonucleotides are immobilized on the control particles, partially immobilized on the control particles, encapsulated in the control particles, partially encapsulated in the control particles, or a combination thereof.

102. A control particle composition according to any one of claims 86 to 101, wherein the control particle is disintegrable.

103. The control particle composition of any one of claims 86 to 102, wherein the control particles comprise beads.

104. 104. The control particle composition of any one of claims 86 to 103, wherein the control particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; or wherein the control particles comprise polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof.

105. The control particle composition of any one of claims 86 to 104, wherein the control particle comprises a collapsible hydrogel particle.

106. A control particle composition according to any one of claims 86 to 105, wherein the control particle is associated with a detectable moiety.

107. 107. The control particle composition of any one of claims 86 to 106, wherein the control particle oligonucleotide is associated with a detectable moiety.

108. the control particles are associated with a plurality of first cellular component binding reagents; A control particle composition according to any one of claims 86 to 107, wherein at least one of the plurality of first cellular component binding reagents is associated with one of the plurality of control particle oligonucleotides.

109. The control particle composition of claim 108, wherein the first cellular component binding reagent comprises a first antibody.

110. A control particle composition according to any one of claims 108 to 109, wherein the control particle oligonucleotide is bound to the first cellular component binding reagent via a linker.

111. A control particle composition described in any one of claims 108 to 110, wherein the first cellular component binding reagent is associated with two or more of the plurality of control particle oligonucleotides having the same control barcode sequence.

112. A control particle composition described in any one of claims 108 to 111, wherein the first cellular component binding reagent is associated with two or more of the plurality of control particle oligonucleotides having different control barcode sequences.

113. A control particle composition according to any one of claims 108 to 112, wherein at least one of the plurality of first cellular component binding reagents is not associated with any of the plurality of control particle oligonucleotides.

114. The control particle composition of claim 113, wherein the first cellular component binding reagent associated with the control particle oligonucleotide and the first cellular component binding reagent not associated with any control particle oligonucleotide are the same cellular component binding reagent.

115. A control particle composition according to any one of claims 108 to 114, wherein the control particle is associated with a plurality of second cellular component binding reagents.

116. The control particle composition of claim 115, wherein at least one of the plurality of second cellular component binding reagents is associated with one of the plurality of control particle oligonucleotides.

117. The control particle composition of claim 116, wherein the control particle oligonucleotide associated with the first cellular component binding reagent and the control particle oligonucleotide associated with the second cellular component binding reagent comprise different control barcode sequences.

118. The control particle composition of claim 116, wherein the first cellular component binding reagent and the second cellular component binding reagent are the same cellular component binding reagent.

119. A control particle composition described in any one of claims 108 to 118, wherein the first cellular component binding reagent is associated with a partner binding reagent, and the first cellular component binding reagent is associated with the control particle using the partner binding reagent.

120. The control particle composition of claim 119, wherein the partner binding reagent comprises a partner antibody.

121. The control particle composition of claim 120, wherein the partner antibody comprises an anti-cat antibody, an anti-chicken antibody, an anti-cow antibody, an anti-dog antibody, an anti-donkey antibody, an anti-goat antibody, an anti-guinea pig antibody, an anti-hamster antibody, an anti-horse antibody, an anti-human antibody, an anti-llama antibody, an anti-monkey antibody, an anti-mouse antibody, an anti-pig antibody, an anti-rabbit antibody, an anti-rat antibody, an anti-sheep antibody, or a combination thereof.

122. The partner antibody may be an immunoglobulin G (IgG), an F(ab') fragment, an F(ab') 2 122. The control particle composition of any one of claims 120 to 121, comprising a fragment thereof, a combination thereof, or a fragment thereof.

123. 123. The control particle composition of any one of claims 108 to 122, wherein the first cellular component binding reagent is associated with a detectable moiety.

124. 124. The control particle composition of any one of claims 108 to 123, wherein the second cellular component binding reagent is associated with a detectable moiety.

125. A control particle composition according to any one of claims 86 to 124; Multiple barcodes and Includes a kit.

126. a barcode of the plurality of barcodes comprising a target binding region and a molecular beacon sequence; 126. The kit of claim 125, wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences.

127. 127. The kit of claim 126, wherein the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

128. 128. The kit of any one of claims 126 to 127, wherein the target binding region comprises a poly(dT) region.

129. 129. The kit of any one of claims 125 to 128, wherein the plurality of barcodes are associated with bar-coding particles.

130. 130. The kit of claim 129, wherein at least one barcode of the plurality of barcodes is immobilized on the bar-coding particle, partially immobilized on the bar-coding particle, encapsulated in the bar-coding particle, partially encapsulated in the bar-coding particle, or a combination thereof.

131. The kit of any one of claims 129 to 130, wherein the bar-coding particles are disintegrating.

132. 1. A method for determining the number of targets, comprising: using the plurality of stochastic barcodes to stochastically barcode a plurality of target and a plurality of control particle oligonucleotides in a cell of the plurality of cells to generate a plurality of stochastically barcoded target and a plurality of stochastically barcoded control particle oligonucleotides; each of the plurality of probabilistic barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two of the plurality of probabilistic barcodes comprise different sequences, and at least two of the plurality of probabilistic barcodes comprise the same cell labeling sequence; a control particle composition comprising control particles associated with said plurality of control particle oligonucleotides, each of said plurality of control particle oligonucleotides comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to at least one target binding region of said plurality of stochastic barcodes; acquiring sequencing data for the plurality of stochastically barcoded target and the plurality of stochastically barcoded control particle oligonucleotides; Counting the number of molecular label sequences having distinguishable sequences associated with the plurality of control particle oligonucleotides having the control barcode sequence in the sequencing data; For at least one target of said plurality of targets: counting the number of molecular beacon sequences having distinguishable sequences associated with the target in the sequencing data; and estimating the number of targets, wherein the estimated number of targets correlates with the number of molecular beacon sequences having distinguishable sequences associated with the counted targets and the number of molecular beacon sequences having distinguishable sequences associated with the control barcode sequences.

133. 133. The method of claim 132, wherein the pseudo-target region comprises a poly(dA) region.

134. 134. The method of any one of claims 132 to 133, wherein the pseudo-target region comprises a subsequence of the target.

135. 135. The method of any one of claims 132 to 134, wherein the control barcode sequence is 6 to 60 nucleotides in length.

136. 136. The method of any one of claims 132-135, wherein the control barcode oligonucleotide is 50-500 nucleotides in length.

137. 137. The method of any one of claims 132-136, wherein the control barcode sequences of at least 5, 10, 100, 1000, or a combination thereof, of the plurality of control particle oligonucleotides are identical.

138. 138. The method of any one of claims 132-137, wherein at least 3, 5, 10, 100, or a combination thereof, of the plurality of control particle oligonucleotides comprise different control barcode sequences.

139. the plurality of control particle oligonucleotides a plurality of first control particle oligonucleotides each comprising a first control barcode sequence; a plurality of second control particle oligonucleotides, each of which comprises a second control barcode sequence; 139. The method of any one of claims 132 to 138, wherein the first control barcode sequence and the second control barcode sequence have different sequences.

140. 140. The method of claim 139, wherein the number of the first control particle oligonucleotides and the number of the second control particle oligonucleotides are approximately the same.

141. 140. The method of claim 139, wherein the number of the first plurality of control particle oligonucleotides and the number of the second plurality of control particle oligonucleotides are different.

142. 142. The method of claim 141, wherein the number of the plurality of first control particle oligonucleotides is at least 2 times, 10 times, 100 times, or a combination thereof, greater than the number of the plurality of second control particle oligonucleotides.

143. Counting the number of molecular label sequences having distinguishable sequences associated with the plurality of control particle oligonucleotides having the control barcode sequence in the sequencing data, counting the number of molecular landmark sequences having identifiable sequences associated with the first control barcode sequence in the sequencing data; and counting the number of molecular landmark sequences having identifiable sequences associated with the second control barcode sequence in the sequencing data.

144. The estimated number of targets is the number of molecular beacon sequences having distinguishable sequences associated with the target counted; the number of molecular beacon sequences having distinguishable sequences associated with the first control barcode sequence; and the number of molecular beacon sequences having distinguishable sequences associated with the second control barcode sequence; The method of claim 143, wherein the correlation is

145. The estimated number of targets is the number of molecular beacon sequences having distinguishable sequences associated with the target counted; the number of molecular beacon sequences having distinguishable sequences associated with the control barcode sequence; and The number of the plurality of control particle oligonucleotides that contain the control barcode sequence The method of any one of claims 132 to 144, wherein the

146. The estimated number of targets is the number of molecular beacon sequences having distinguishable sequences associated with the target that were counted; and the number of the plurality of control particle oligonucleotides comprising the control barcode sequence; and the number of molecular beacon sequences having distinguishable sequences associated with the control barcode sequence; The method of claim 145, wherein the ratio is correlated to:

147. 147. The method of any one of claims 132 to 146, wherein the control particle oligonucleotide is non-homologous to the genomic sequence of the cell, non-homologous to the genomic sequence of a species, homologous to the genomic sequence of a species, or a combination thereof.

148. 148. The method of claim 147, wherein the species is a non-mammalian species.

149. 149. The method of any one of claims 132 to 148, wherein the control particle oligonucleotide is attached to the control particle via a linker.

150. 150. The method of any one of claims 132-149, wherein the control particle has a diameter of about 1-1000 micrometers, about 10-100 micrometers, about 7.5 micrometers, or a combination thereof.

151. The method of any one of claims 132 to 150, wherein the plurality of control particle oligonucleotides are immobilized on the control particles, partially immobilized on the control particles, encapsulated in the control particles, partially encapsulated in the control particles, or a combination thereof.

152. 152. The method of any one of claims 132 to 151, comprising releasing at least one of the plurality of control particle oligonucleotides from the control particles prior to the step of stochastically barcoding the plurality of target and control particles and the plurality of control particle oligonucleotides.

153. 153. The method of any one of claims 132 to 152, wherein the control particles are disintegrating.

154. The method of any one of claims 132 to 153, wherein the control particles comprise control particle beads.

155. 155. The method of any one of claims 132-154, wherein the control particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; or wherein the control particles comprise polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof.

156. The method of any one of claims 132 to 155, wherein the control particles comprise collapsible hydrogel particles.

157. 157. The method of any one of claims 132 to 156, wherein the control particle is associated with a detectable moiety.

158. 158. The method of any one of claims 132 to 157, wherein the control particle oligonucleotide is associated with a detectable moiety.

159. the control particles are associated with a plurality of first cellular component binding reagents; 159. The method of any one of claims 132 to 158, wherein at least one of the plurality of first cellular component binding reagents is associated with one of the plurality of control particle oligonucleotides.

160. 160. The method of claim 159, wherein the first cellular component binding reagent comprises a first antibody.

161. 161. The method of any one of claims 159 to 160, wherein the control particle oligonucleotide is attached to the first cellular component binding reagent via a linker.

162. 162. The method of any one of claims 159 to 161, wherein the first cellular component binding reagent is associated with two or more of the plurality of control particle oligonucleotides having the same control barcode sequence.

163. 163. The method of any one of claims 159 to 162, wherein the first cellular component binding reagent is associated with two or more of the plurality of control particle oligonucleotides having different control barcode sequences.

164. 164. The method of any one of claims 159 to 163, wherein at least one of the plurality of first cellular component binding reagents is not associated with any of the plurality of control particle oligonucleotides.

165. The method of claim 164, wherein the first cellular component binding reagent associated with the control particle oligonucleotide and the first cellular component binding reagent not associated with any control particle oligonucleotide are the same cellular component binding reagent.

166. 166. The method of any one of claims 164 to 165, wherein the control particles are associated with a plurality of second cellular component binding reagents.

167. 167. The method of claim 166, wherein at least one of the plurality of second cellular component binding reagents is associated with one of the plurality of control particle oligonucleotides.

168. The method of claim 167, wherein the control particle oligonucleotide associated with the first cellular component binding reagent and the control particle oligonucleotide associated with the second cellular component binding reagent comprise different control barcode sequences.

169. 168. The method of claim 167, wherein the first cellular component binding reagent and the second cellular component binding reagent are the same cellular component binding reagent.

170. 170. The method of any one of claims 159 to 169, wherein the first cellular component binding reagent is associated with a partner binding reagent, and the first cellular component binding reagent is associated with the control particle using the partner binding reagent.

171. 171. The method of claim 170, wherein the partner binding reagent comprises a partner antibody.

172. 172. The method of claim 171, wherein the partner antibody comprises an anti-cat antibody, an anti-chicken antibody, an anti-cow antibody, an anti-dog antibody, an anti-donkey antibody, an anti-goat antibody, an anti-guinea pig antibody, an anti-hamster antibody, an anti-horse antibody, an anti-human antibody, an anti-llama antibody, an anti-monkey antibody, an anti-mouse antibody, an anti-pig antibody, an anti-rabbit antibody, an anti-rat antibody, an anti-sheep antibody, or a combination thereof.

173. The partner antibody may be an immunoglobulin G (IgG), an F(ab') fragment, an F(ab') 2 173. The method of any one of claims 171 to 172, comprising a fragment thereof, a combination thereof, or a fragment thereof.

174. 174. The method of any one of claims 159 to 173, wherein the first cellular component binding reagent is associated with a detectable moiety.

175. 175. The method of any one of claims 159 to 174, wherein the second cellular component binding reagent is associated with a detectable moiety, a binding site for a universal primer, or both.

176. 176. The method of any one of claims 132 to 175, wherein the target binding region comprises a poly(dT) region.

177. 177. The method of any one of claims 132 to 176, wherein the plurality of probabilistic barcodes are associated with bar-coding particles.

178. 178. The method of claim 177, wherein at least one probabilistic barcode of the plurality of probabilistic barcodes is immobilized on the bar-coding particle, partially immobilized on the bar-coding particle, encapsulated in the bar-coding particle, partially encapsulated in the bar-coding particle, or a combination thereof.

179. 179. The method of any one of claims 177 to 178, wherein the bar-coding particles are disintegrating.

180. 180. The method of any one of claims 177 to 179, wherein the bar-coding particles comprise bar-coding beads.

181. 181. The method of any one of claims 177-180, wherein the bar-coding beads comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof, or wherein the bar-coding particles comprise the following materials: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof.

182. 182. The method of any one of claims 177-181, wherein the bar-coding particles comprise collapsible hydrogel particles.

183. 183. The method of any one of claims 177-182, wherein the probabilistic barcode of the bar-coding particle comprises a molecular landmark sequence selected from at least 1,000, 10,000, or a combination thereof, different molecular landmark sequences.

184. 184. The method of any one of claims 177 to 183, wherein the molecular marker sequence of the probabilistic barcode comprises a random sequence.

185. 185. The method of any one of claims 177 to 184, wherein the bar-coding particles comprise at least 10,000 probabilistic barcodes.

186. using the plurality of probabilistic barcodes to probabilistically barcode the plurality of target and control particle oligonucleotides, contacting the plurality of stochastic barcodes with targets of the plurality of targets and control particle oligonucleotides of the plurality of control particle oligonucleotides to generate stochastic barcodes hybridized to the targets and the control particle oligonucleotides; and extending the stochastic barcodes hybridized to the target and control particle oligonucleotides to generate the plurality of stochastic barcoded targets and the plurality of stochastic barcoded control particle oligonucleotides.

187. 187. The method of claim 186, wherein extending the probabilistic barcode comprises extending the probabilistic barcode using a DNA polymerase, a reverse transcriptase, or a combination thereof.

188. 188. The method of any one of claims 132-187, comprising amplifying the plurality of stochastically barcoded target and the plurality of stochastically barcoded control particle oligonucleotides to generate a plurality of amplicons.

189. amplifying the plurality of stochastically barcoded target and the plurality of stochastically barcoded control particle oligonucleotides using polymerase chain reaction (PCR), at least a portion of the molecular beacon sequence and at least a portion of the control particle oligonucleotide; or At least a portion of the molecular beacon sequence and at least a portion of the control particle oligonucleotide The method of claim 188, comprising a step of amplifying the

190. 190. The method of any one of claims 188 to 189, wherein obtaining sequencing data comprises obtaining sequencing data for the plurality of amplicons.

191. The step of obtaining sequencing data comprises: at least a portion of the molecular beacon sequence and at least a portion of the control particle oligonucleotide; or At least a portion of the molecular beacon sequence and at least a portion of the control particle oligonucleotide 191. The method of claim 190, comprising sequencing the

192. 1. A method for cell identification, comprising: contacting a first plurality of cells and a second plurality of cells with two sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets; each of the two sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; barcoding said sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; identifying cell-marker sequences associated with two or more sample-indexed sequences in the obtained sequencing data; and removing sequencing data associated with the cell marker sequence from the obtained sequencing data.

193. contacting the first plurality of cells and the second plurality of cells with the two sample-indexing compositions, respectively; contacting the first plurality of cells with a first sample-indexing composition of the two sample-indexing compositions; and contacting said first plurality of cells with a second sample indexing composition of said two sample indexing compositions.

194. 194. The method of any one of claims 192 to 193, wherein the sample index addition sequence is 6560 nucleotides in length.

195. 195. The method of any one of claims 192 to 194, wherein the sample-indexing oligonucleotide is 50 to 500 nucleotides in length.

196. 196. The method of any one of claims 192-195, wherein the sample indexing sequences of at least 10, 100, 1000, or a combination thereof, of the plurality of sample indexing compositions comprise different sequences.

197. 197. The method of any one of claims 192 to 196, wherein the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof.

198. 200. The method of any one of claims 192 to 197, wherein the sample-indexing oligonucleotide is attached to the cellular component binding reagent via a linker.

199. 200. The method of any one of claims 192 to 198, wherein at least one sample of the plurality of samples comprises a single cell.

200. 200. The method of any one of claims 192 to 199, wherein at least one of the one or more cellular component targets is on a cell surface.

201. 201. A method according to any one of claims 192 to 200, comprising removing any unbound sample indexing composition of the two sample indexing compositions.

202. 202. The method of claim 201, wherein removing the unbound sample-indexing composition comprises washing the cells of the first plurality of cells and the second plurality of cells with a wash buffer.

203. 202. The method of claim 201, wherein the step of removing the unbound sample indexing compositions comprises using flow cytometry to select cells bound to a cellular component binding reagent of at least one of the two sample indexing compositions.

204. 204. The method of any one of claims 192 to 203, comprising lysing the one or more cells from each of the plurality of samples.

205. 205. The method of any one of claims 192 to 204, wherein the sample-indexing oligonucleotide is configured to be detachable or non-detachable from the cellular component binding reagent.

206. 206. The method of any one of claims 192 to 205, comprising the step of cleaving the sample-indexing oligonucleotide from the cellular component binding reagent.

207. 207. The method of claim 206, wherein the step of cleaving the sample index-added oligonucleotide comprises cleaving the sample index-added oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof.

208. 208. The method of any one of claims 192 to 207, wherein the sample-indexing oligonucleotide is not homologous to any genomic sequence of the one or more cells, or is homologous to a genomic sequence of a species, or a combination thereof.

209. 209. The method of claim 208, wherein the species is a non-mammalian species.

210. 210. The method of any one of claims 192 to 209, wherein one sample of the plurality of samples comprises a plurality of cells, tissues, tumor samples, or any combination thereof.

211. 211. The method of any one of claims 192-210, wherein the plurality of samples comprises mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof.

212. 212. The method of any one of claims 192-211, wherein the sample-indexing oligonucleotide comprises a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes.

213. 213. The method of claim 212, wherein the barcode comprises a target binding region that includes the capture sequence.

214. 214. The method of claim 213, wherein the target binding region comprises a poly(dT) region.

215. 215. The method of any one of claims 212 to 214, wherein the sequence of the sample-indexing oligonucleotide complementary to the capture sequence of the barcode comprises a poly(dA) region.

216. 216. The method of any one of claims 192-215, wherein the sample-indexing oligonucleotide comprises a molecular beacon sequence, a binding site for a universal primer, or both.

217. 217. The method of any one of claims 192-216, wherein the cellular component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

218. 218. The method of any one of claims 192 to 217, wherein the cellular component target is selected from a group comprising 10 to 100 different protein targets.

219. 219. The method of any one of claims 192-218, wherein the cellular component binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence.

220. 219. The method of any one of claims 192 to 218, wherein the cellular component binding reagent is associated with two or more sample indexing oligonucleotides having different sample indexing sequences.

221. The method of any one of claims 192 to 220, wherein the sample indexing composition of the plurality of sample indexing compositions comprises a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide.

222. 222. The method of claim 221, wherein the cellular component binding reagent and the second cellular component binding reagent are the same.

223. 223. The method of any one of claims 192 to 222, wherein the cellular component binding reagent is associated with a detectable moiety.

224. a barcode of the plurality of barcodes comprising a target binding region and a molecular beacon sequence; 224. The method of any one of claims 192 to 223, wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences.

225. 225. The method of claim 224, wherein the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

226. 226. The method of any one of claims 224 to 225, wherein the target binding region comprises a poly(dT) region.

227. 227. The method of any one of claims 224 to 226, wherein the plurality of barcodes are associated with particles.

228. 228. The method of claim 227, wherein at least one barcode of the plurality of barcodes is immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or a combination thereof.

229. 229. The method of any one of claims 227 to 228, wherein the particles are disintegrating.

230. 230. The method of any one of claims 227 to 229, wherein the particles comprise beads.

231. 231. The method of any one of claims 227-230, wherein the beads comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof, or wherein the particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

232. 232. The method of any one of claims 227 to 231, wherein the particles comprise collapsible hydrogel particles.

233. 233. The control particle composition of any one of claims 227 to 232, wherein the particle is associated with a detectable moiety.

234. 233. The control particle composition of any one of claims 227-232, wherein the sample-indexing oligonucleotide is associated with a detectable moiety.

235. 235. The method of any one of claims 227-234, wherein the barcode of the particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or a combination thereof, different molecular beacon sequences.

236. 236. The method of claim 235, wherein the molecular marker sequence of the barcode comprises a random sequence.

237. 237. The method of any one of claims 227 to 236, wherein the particles comprise at least 10,000 barcodes.

238. barcoding said sample-indexed oligonucleotides using said plurality of barcodes, contacting the plurality of barcodes with the sample-indexed oligonucleotides to produce barcodes hybridized to the sample-indexed oligonucleotides; and extending the barcode hybridized to the sample-indexed oligonucleotide to generate the plurality of barcoded sample-indexed oligonucleotides.

239. 239. The method of claim 238, wherein extending the barcodes comprises using a DNA polymerase to extend the barcodes to generate the plurality of barcoded sample-indexed oligonucleotides.

240. 239. The method of claim 238, wherein extending the barcodes comprises using a reverse transcriptase to extend the barcodes to generate the plurality of barcoded sample-indexed oligonucleotides.

241. 241. The method of any one of claims 238-240, comprising amplifying said plurality of barcoded, sample-indexed oligonucleotides to generate a plurality of amplicons.

242. 242. The method of claim 241, wherein amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides using polymerase chain reaction (PCR).

243. 243. The method of any one of claims 241-242, wherein obtaining the sequencing data for the plurality of barcoded, sample-indexed oligonucleotides comprises obtaining sequencing data for the plurality of amplicons.

244. 244. The method of Claim 243, wherein obtaining said sequencing data comprises sequencing at least a portion of said molecular beacon sequence and at least a portion of said sample-indexing oligonucleotides.

245. 245. The method of any one of claims 192 to 244, wherein identifying the sample origin of the at least one cell comprises identifying the sample origin of the plurality of barcoded targets based on the sample indexing sequence of the at least one barcoded sample indexing oligonucleotide.

246. 246. The method of any one of claims 192 to 245, wherein barcoding the sample-indexed oligonucleotides using the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides using a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

247. barcoding a plurality of targets of the cells using the plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell-labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell-labeling sequence; and obtaining sequencing data for the barcoded target.

248. barcoding the plurality of targets using the plurality of barcodes to generate the plurality of barcoded targets; contacting a copy of the target with the target binding region of the barcode; and reverse transcribing said plurality of targets using said plurality of barcodes to generate a plurality of reverse transcribed targets.

249. 249. The method of any one of claims 247 to 248, comprising amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining the sequencing data for the plurality of barcoded targets.

250. 250. The method of claim 249, wherein amplifying the barcoded target to generate the plurality of amplified barcoded targets comprises amplifying the barcoded target by polymerase chain reaction (PCR).

251. 251. The method of any one of claims 247 to 250, wherein barcoding the plurality of targets of the cells using the plurality of barcodes to generate the plurality of barcoded targets comprises probabilistically barcoding the plurality of targets of the cells using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

252. 1. A method for sequencing control, comprising: contacting one or more cells of the plurality of cells with a control composition of a plurality of control compositions; a cell of the plurality of cells comprises a plurality of targets and a plurality of protein targets; each of the plurality of control compositions comprising a protein binding reagent associated with a control oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the plurality of protein targets, the control oligonucleotide comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to the target binding region of at least one of the plurality of barcodes; barcoding said control oligonucleotides with a plurality of barcodes to generate a plurality of barcoded control oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded control oligonucleotides; and determining at least one characteristic of the one or more cells using at least one characteristic of the plurality of barcoded control oligonucleotides in the sequencing data.

253. 1. A method for sequencing control, comprising: contacting one or more cells of the plurality of cells with a control composition of a plurality of control compositions; a cell of the plurality of cells comprises a plurality of targets and a plurality of cellular component targets; each of the plurality of control compositions comprises a cellular component binding reagent associated with a control oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the plurality of cellular component targets, the control oligonucleotide comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to the target binding region of at least one of the plurality of barcodes; barcoding said control oligonucleotides with a plurality of barcodes to generate a plurality of barcoded control oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded control oligonucleotides; and determining at least one characteristic of the one or more cells using at least one characteristic of the plurality of barcoded control oligonucleotides in the sequencing data.

254. 254. The method of claim 253, wherein the cellular component target comprises a protein target.

255. 1. A method for sequencing control, comprising: contacting one or more cells of the plurality of cells with a control composition of a plurality of control compositions; a cell of the plurality of cells comprises a plurality of targets and a plurality of cellular component targets; each of the plurality of control compositions comprises a cellular component binding reagent associated with a control oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the plurality of cellular component targets, the control oligonucleotide comprising a control barcode sequence and a pseudo target region comprising a sequence substantially complementary to the target binding region of at least one of the plurality of barcodes; and determining at least one characteristic of said one or more cells using at least one characteristic of said plurality of control oligonucleotides.

256. 256. The method of claim 255, wherein the cellular component target comprises a protein target.

257. barcoding the control oligonucleotide using a plurality of barcodes to generate a plurality of barcoded control oligonucleotides, each of the plurality of barcodes comprising a cell label sequence, a molecular label sequence, and a target binding region, the molecular label sequences of at least two barcodes of the plurality of barcodes comprising different sequences, and at least two barcodes of the plurality of barcodes comprising the same cell label sequence; and obtaining sequencing data for the plurality of barcoded control oligonucleotides.

258. 258. The method of any one of claims 252 to 257, wherein the pseudo-target region comprises a poly(dA) region.

259. 259. The method of any one of claims 252 to 258, wherein the control barcode sequence is 6 to 60 nucleotides in length.

260. 260. The method of any one of claims 252-259, wherein the control barcode oligonucleotide is 50-500 nucleotides in length.

261. 261. The method of any one of claims 252-260, wherein the control barcode sequences of at least 2, 10, 100, 1000, or a combination thereof, of the plurality of control particle oligonucleotides are identical.

262. 262. The method of any one of claims 252-261, wherein at least 2, 10, 100, 1000, or a combination thereof, of the plurality of control particle oligonucleotides comprise different control barcode sequences.

263. determining said at least one characteristic of said one or more cells determining the number of cell labeling sequences having distinguishable sequences associated with the plurality of barcoded control oligonucleotides in the sequencing data; and determining the number of said one or more cells using a number of cell labeling sequences having distinguishable sequences associated with said plurality of barcoded control oligonucleotides.

264. 264. The method of claim 263, comprising determining a single cell capture efficiency based on the determined number of the one or more cells.

265. The method of claim 263, comprising determining a single cell capture efficiency based on the ratio of the determined number of the one or more cells to the number of the plurality of cells.

266. determining said at least one characteristic of said one or more cells determining, for each cell label in the sequencing data, the number of molecular label sequences having distinguishable sequences associated with the cell label and the control barcode sequence; and determining the number of said one or more cells using a number of molecular label sequences having distinguishable sequences associated with said cell label and said control barcode sequence.

267. determining the number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence; 267. The method of claim 266, comprising determining, for each cell marker in the sequencing data, the number of molecular marker sequences having the greatest number of distinguishable sequences associated with the cell marker and the control barcode sequence.

268. determining the number of the one or more cells using a number of molecular marker sequences having distinguishable sequences associated with the cell marker and the control barcode sequence; generating a plot of the number of cellular markers and the number of molecular marker sequences with the most distinguishable sequences in the sequencing data associated with the number of molecular marker sequences with the most distinguishable sequences; and determining a cutoff in the plot as the number of said one or more cells.

269. 269. The method of any one of claims 252-268, wherein the control oligonucleotide is not homologous to any genomic sequence of any of the plurality of cells, or wherein the control oligonucleotide is homologous to a genomic sequence of one species.

270. 270. The method of claim 269, wherein the species is a non-mammalian species.

271. 271. The method of any one of claims 252 to 270, comprising releasing the control oligonucleotide from the protein-binding reagent or the cellular component-binding reagent prior to barcoding the control oligonucleotide.

272. 272. The method of any one of claims 252 to 271, comprising the step of removing unbound control compositions from the plurality of control compositions.

273. 273. The method of claim 272, wherein the step of removing the unbound control composition comprises washing the one or more cells of the plurality of cells with a wash buffer.

274. The method of claim 273, wherein the step of removing the unbound sample indexing composition comprises using flow cytometry to select cells bound to at least one protein binding reagent or at least one cellular component binding reagent of the control composition.

275. 275. The method of any one of claims 252-274, wherein at least one of said plurality of protein targets or at least one of said plurality of cellular component targets is expressed on the cell surface.

276. 276. The method of any one of claims 252-275, wherein at least one of said plurality of protein targets or at least one of said plurality of cellular component targets comprises a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof.

277. 277. The method of any one of claims 252 to 276, wherein the protein-binding reagent or the cellular component-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, a filtrate, or a combination thereof.

278. The method of any one of claims 252 to 277, wherein the protein target of the protein-binding reagent is selected from a group comprising 10 to 100 different protein targets, or the cellular component target of the cellular component-binding reagent is selected from a group comprising 10 to 100 different cellular component targets.

279. The method of claim 278, wherein the protein target of the protein binding reagent or the cellular component target of the cellular component binding reagent comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, an intracellular protein, or any combination thereof.

280. 280. The method of any one of claims 252 to 279, wherein the control oligonucleotide is attached to the protein binding reagent or the cellular component binding reagent via a linker.

281. 281. The method of any one of claims 252 to 280, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more control oligonucleotides having the same control barcode sequence.

282. 282. The method of any one of claims 252 to 281, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more control oligonucleotides having different but identical control barcode sequences.

283. 283. The method of any one of claims 252-282, wherein the protein-binding reagent or the second cellular component-binding reagent of said plurality of control compositions is not associated with said control oligonucleotide.

284. 284. The method of claim 283, wherein the protein binding reagent and the second protein binding reagent are the same, or the cellular component binding reagent and the second cellular component binding reagent are the same.

285. 285. The method of any one of claims 252 to 284, wherein the barcode comprises a binding site for a universal primer.

286. 286. The method of any one of claims 252 to 285, wherein the target binding region comprises a poly(dT) region.

287. 287. The method of any one of claims 252 to 286, wherein the plurality of barcodes are associated with bar-coding particles.

288. 288. The method of claim 287, wherein at least one barcode of the plurality of barcodes is immobilized on the bar-coding particle, partially immobilized on the bar-coding particle, encapsulated in the bar-coding particle, partially encapsulated in the bar-coding particle, or a combination thereof.

289. The method of any one of claims 287 to 288, wherein the bar-coding particles are disintegrating.

290. 290. The method of any one of claims 287 to 289, wherein the bar-coding particles comprise bar-coding beads.

291. 291. The method of any one of claims 287-290, wherein the bar-coded particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; or wherein the bar-coded particles comprise the following materials: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof.

292. 292. The method of any one of claims 287-291, wherein the bar-coding particles comprise collapsible hydrogel particles.

293. 293. The method of any one of claims 287 to 292, wherein the bar-coding particle is associated with an optical moiety.

294. The control particle composition of any one of claims 287 to 293, wherein the control oligonucleotide is associated with an optical moiety.

295. 295. The method of any one of claims 287-294, wherein the barcode of the bar-coding particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or a combination thereof, different molecular beacon sequences.

296. 296. The method of any one of claims 287 to 295, wherein the molecular beacon sequence of the barcode comprises a random sequence.

297. 297. The method of any one of claims 287 to 296, wherein the bar-coding particles comprise at least 10,000 barcodes.

298. 300. The method of any one of claims 252 to 297, wherein barcoding the control oligonucleotides comprises barcoding the control oligonucleotides with a plurality of stochastic barcodes to generate a plurality of stochastically barcoded control oligonucleotides.

299. barcoding the plurality of control oligonucleotides using the plurality of barcodes, contacting the plurality of barcodes with control oligonucleotides of the plurality of control compositions to produce barcodes hybridized to the control oligonucleotides; and extending the probability barcodes hybridized to the control oligonucleotides to generate the plurality of barcoded control oligonucleotides.

300. 300. The method of claim 299, wherein extending the barcode comprises extending the barcode using a DNA polymerase, a reverse transcriptase, or a combination thereof.

301. 301. The method of any one of claims 252-300, comprising amplifying said plurality of barcoded control oligonucleotides to generate a plurality of amplicons.

302. 302. The method of claim 301, wherein amplifying the plurality of barcoded control oligonucleotides comprises amplifying at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotides using polymerase chain reaction (PCR).

303. 303. The method of any one of claims 301 to 302, wherein said obtaining sequencing data comprises obtaining sequencing data for said plurality of amplicons.

304. 304. The method of claim 303, wherein obtaining the sequencing data comprises sequencing at least a portion of the molecular beacon sequence and at least a portion of the control oligonucleotide.

305. 1. A method for cell identification, comprising: contacting a first plurality of cells and a second plurality of cells with two sample-indexing compositions of the plurality of sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more protein targets; each of the two sample indexing compositions comprises a protein binding reagent associated with a sample indexing oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the one or more protein targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; barcoding said sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; identifying one or more cell-marker sequences each associated with two or more sample-indexed sequences in the obtained sequencing data; and removing from the obtained sequencing data the sequencing data associated with the one or more cell labeling sequences, each associated with two or more sample indexing sequences.

306. 1. A method for identification of multiplets, comprising: contacting a first plurality of cells and a second plurality of cells with two sample-indexing compositions of the plurality of sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more protein targets; each of the two sample indexing compositions comprises a protein binding reagent associated with a sample indexing oligonucleotide, the protein binding reagent capable of specifically binding to at least one of the one or more protein targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; barcoding said sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; and identifying one or more multicellular label sequences each associated with two or more sample-indexed sequences in the obtained sequencing data.

307. 1. A method for cell identification, comprising: contacting a first plurality of cells and a second plurality of cells with two sample-indexing compositions of the plurality of sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets; each of the two sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; barcoding said sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; identifying one or more cell-marker sequences each associated with two or more sample-indexed sequences in the obtained sequencing data; and removing from the obtained sequencing data the sequencing data associated with the one or more cell labeling sequences, each associated with two or more sample indexing sequences.

308. 1. A method for identification of multiplets, comprising: contacting a first plurality of cells and a second plurality of cells with two sample-indexing compositions of the plurality of sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets; each of the two sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; barcoding said sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; and identifying one or more multicellular label sequences each associated with two or more sample-indexed sequences in the sequencing data.

309. 309. The method of any one of claims 307-308, wherein the cellular component binding reagent comprises a protein binding reagent, the protein binding reagent is associated with the sample-indexing oligonucleotide, and the one or more cellular component targets comprise one or more protein targets.

310. contacting the first plurality of cells and the second plurality of cells with the two sample-indexing compositions, respectively; contacting the first plurality of cells with a first sample-indexing composition of the two sample-indexing compositions; and contacting said first plurality of cells with a second sample indexing composition of said two sample indexing compositions.

311. 1. A method for sample identification, comprising: contacting one or more cells from each of the first plurality of cells and the second plurality of cells with a sample-indexing composition of a plurality of two sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets; each of the two sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; and identifying one or more cells each associated with two or more sample-indexing sequences.

312. 1. A method for identification of multiplets, comprising: contacting one or more cells from each of the first plurality of cells and the second plurality of cells with a sample-indexing composition of a plurality of two sample-indexing compositions, respectively; each of the first plurality of cells and each of the second plurality of cells comprises one or more cellular component targets; each of the two sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotide comprises a sample indexing sequence, and the sample indexing sequences of the two sample indexing compositions comprise different sequences; and identifying one or more cells each associated with two or more sample-indexed sequences as multicellular.

313. contacting the first plurality of cells and the second plurality of cells with the two sample-indexing compositions, respectively; contacting the first plurality of cells with a first sample-indexing composition of the two sample-indexing compositions; and contacting said first plurality of cells with a second sample indexing composition of said two sample indexing compositions.

314. identifying the one or more cells each associated with two or more sample-indexing sequences, barcoding said sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; each of the plurality of barcodes comprises a cell labeling sequence, a molecular labeling sequence, and a target binding region, the molecular labeling sequences of at least two barcodes of the plurality of barcodes comprise different sequences, and at least two barcodes of the plurality of barcodes comprise the same cell labeling sequence; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; and identifying one or more cell marker sequences each associated with two or more sample-indexed sequences in the obtained sequencing data.

315. 315. The method of claim 314, comprising removing from the obtained sequencing data the sequencing data associated with the one or more cell marker sequences, each associated with two or more sample-indexing sequences.

316. 315. The method of claim 314, comprising excluding from subsequent analysis the sequencing data associated with the one or more cell marker sequences each associated with the two or more sample-indexing sequences.

317. 317. The method of any one of claims 305 to 316, wherein the sample index addition sequence is 6 to 60 nucleotides in length.

318. 318. The method of any one of claims 305-317, wherein the sample-indexing oligonucleotide is 50-500 nucleotides in length.

319. 319. The method of any one of claims 305-318, wherein the sample indexing sequences of at least 10, 100, 1000, or a combination thereof, of the plurality of sample indexing compositions comprise different sequences.

320. 320. The method of any one of claims 305 to 319, wherein the protein binding reagent or the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, an integrin, or a combination thereof.

321. 321. The method of any one of claims 305 to 320, wherein the sample-indexing oligonucleotide is attached to the protein-binding reagent or the cellular component-binding reagent via a linker.

322. 322. The method of any one of claims 305-321, wherein at least one of the first plurality of cells and the second plurality of cells comprises a single cell.

323. 323. The method of any one of claims 305-322, wherein at least one of said at least one or more protein targets or said one or more cellular component targets is on a cell surface.

324. 324. The method of any one of claims 305 to 323, comprising removing unbound sample indexing compositions of the two sample indexing compositions.

325. 325. The method of claim 324, wherein removing the unbound sample-indexing composition comprises washing the cells of the first plurality of cells and the second plurality of cells with a wash buffer.

326. The method of claim 324, wherein the step of removing the unbound sample indexing compositions comprises using flow cytometry to select cells bound to at least one protein binding reagent or at least one cellular component binding reagent of the two sample indexing compositions.

327. 327. The method of any one of claims 305 to 326, comprising lysing cells of the first plurality of cells and the second plurality of cells.

328. 328. The method of any one of claims 305 to 327, wherein the sample-indexing oligonucleotide is configured to be detachable or non-detachable from the protein-binding reagent or the cellular component-binding reagent.

329. 329. The method of any one of claims 305 to 328, comprising the step of cleaving the sample-indexing oligonucleotide from the protein-binding reagent or the cellular component-binding reagent.

330. 330. The method of claim 329, wherein the step of cleaving the sample index-added oligonucleotide comprises cleaving the sample index-added oligonucleotide from the protein binding reagent or the cellular component binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof.

331. 331. The method of any one of claims 305-330, wherein said sample-indexing oligonucleotide is not homologous to any genomic sequence of said one or more cells.

332. 332. The method of any one of claims 305-331, wherein the sample-indexing oligonucleotide is homologous to a genomic sequence of a species.

333. 333. The method of any one of claims 305-332, wherein at least one of the first plurality of cells and the second plurality of cells comprises a tumor cell or a non-tumor cell.

334. 334. The method of any one of claims 305-333, wherein at least one of the first plurality of cells and the second plurality of cells comprises mammalian cells, bacterial cells, viral cells, yeast cells, fungal cells, or any combination thereof.

335. 335. The method of any one of claims 305-334, wherein the sample-indexing oligonucleotide comprises a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes.

336. 336. The method of claim 335, wherein the barcode comprises a target binding region comprising the capture sequence.

337. 337. The method of claim 336, wherein the target binding region comprises a poly(dT) region.

338. 338. The method of any one of claims 305-337, wherein the sequence of the sample-indexing oligonucleotide complementary to the capture sequence of the barcode comprises a poly(dA) region.

339. 339. The method of any one of claims 305-338, wherein the sample-indexing oligonucleotide comprises a molecular beacon sequence, a binding site for a universal primer, or both.

340. 340. The method of any one of claims 305-339, wherein the protein target or the cellular component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

341. 341. The method of any one of claims 305 to 340, wherein the protein target is selected from a group comprising 10 to 100 different protein targets, or the cellular component target is selected from a group comprising 10 to 100 different cellular component targets.

342. 342. The method of any one of claims 305-341, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence.

343. 342. The method of any one of claims 305 to 341, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more sample-indexing oligonucleotides having different sample-indexing sequences.

344. The method of any one of claims 305 to 343, wherein the sample indexing composition of the plurality of sample indexing compositions comprises a second protein binding reagent or a second cellular component binding reagent that is not bound to the sample indexing oligonucleotide.

345. The method of claim 344, wherein the protein binding reagent and the second protein binding reagent are the same, or the cellular component binding reagent and the second cellular component binding reagent are the same.

346. 346. The method of any one of claims 305 to 345, wherein the protein-binding reagent or the cellular component-binding reagent is associated with a detectable moiety.

347. a barcode of the plurality of barcodes comprising a target binding region and a molecular beacon sequence; The method of any one of claims 305 to 346, wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences.

348. The method of claim 347, wherein the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

349. The method of any one of claims 347 to 348, wherein the target binding region comprises a poly(dT) region.

350. 350. The method of any one of claims 347 to 349, wherein the plurality of barcodes are associated with particles.

351. 351. The method of claim 350, wherein at least one barcode of the plurality of barcodes is immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or a combination thereof.

352. 352. The method of any one of claims 350 to 351, wherein the particles are disintegrating.

353. 353. The method of any one of claims 350 to 352, wherein the particles comprise beads.

354. 354. The method of any one of claims 350-353, wherein the particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; or wherein the particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogels, paramagnetic materials, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

355. The method of any one of claims 350 to 354, wherein the particles comprise collapsible hydrogel particles.

356. 356. The method of any one of claims 350 to 355, wherein the particle is associated with a detectable moiety.

357. 357. The method of any one of claims 350 to 356, wherein the sample-indexing oligonucleotide is associated with a detectable moiety.

358. 358. The method of any one of claims 350-357, wherein the barcode of the particle comprises a molecular beacon sequence selected from at least 1,000, 10,000, or a combination thereof, different molecular beacon sequences.

359. 359. The method of any one of claims 350 to 358, wherein the molecular beacon sequence of the barcode comprises a random sequence.

360. 360. The method of any one of claims 350 to 359, wherein the particles comprise at least 10,000 barcodes.

361. barcoding said sample-indexed oligonucleotides using said plurality of barcodes, contacting the plurality of barcodes with the sample-indexed oligonucleotides to produce barcodes hybridized to the sample-indexed oligonucleotides; and extending the barcode hybridized to the sample-indexed oligonucleotide to generate the plurality of barcoded sample-indexed oligonucleotides.

362. 362. The method of claim 361, wherein extending the barcode comprises using a DNA polymerase to extend the barcode to generate the plurality of barcoded sample-indexed oligonucleotides.

363. 362. The method of claim 361, wherein extending the barcodes comprises using a reverse transcriptase to extend the barcodes to generate the plurality of barcoded sample-indexed oligonucleotides.

364. 364. The method of any one of claims 361-363, comprising amplifying said plurality of barcoded, sample-indexed oligonucleotides to generate a plurality of amplicons.

365. 365. The method of claim 364, wherein amplifying the plurality of barcoded sample indexing oligonucleotides comprises amplifying at least a portion of the molecular label sequence and at least a portion of the sample indexing oligonucleotides using polymerase chain reaction (PCR).

366. 366. The method of any one of claims 364-365, wherein obtaining said sequencing data for said plurality of barcoded, sample-indexed oligonucleotides comprises obtaining sequencing data for said plurality of amplicons.

367. 367. The method of claim 366, wherein obtaining the sequencing data comprises sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotide.

368. 368. The method of any one of claims 305-367, wherein barcoding the sample-indexed oligonucleotides using the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides using a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

369. barcoding a plurality of targets of the cells using the plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell-labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell-labeling sequence; and obtaining sequencing data for said barcoded targets.

370. barcoding the plurality of targets using the plurality of barcodes to generate the plurality of barcoded targets; contacting a copy of the target with the target binding region of the barcode; and reverse transcribing said plurality of targets using said plurality of barcodes to generate a plurality of reverse transcribed targets.

371. 371. The method of any one of claims 369-370, comprising amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining the sequencing data for the plurality of barcoded targets.

372. 372. The method of claim 371, wherein amplifying the barcoded target to generate the plurality of amplified barcoded targets comprises amplifying the barcoded target by polymerase chain reaction (PCR).

373. 373. The method of any one of claims 369-372, wherein barcoding the plurality of targets of the cells using the plurality of barcodes to generate the plurality of barcoded targets comprises probabilistically barcoding the plurality of targets of the cells using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

374. 1. A method for determining protein-protein interactions, comprising: contacting the cell with a first pair of interaction determining compositions, the cell comprises a first protein target and a second protein target; each of said first pair of interaction-determining compositions comprises a protein binding reagent associated with an interaction-determining oligonucleotide, said protein binding reagent of one of said first pair of interaction-determining compositions being capable of specifically binding to said first protein target and said protein binding reagent of the other of said first pair of interaction-determining compositions being capable of specifically binding to said second protein target; the interaction determining oligonucleotides comprise an interaction determining sequence and a bridging oligonucleotide hybridization region, and the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; ligating the interaction determining oligonucleotides of the first pair of interaction determining compositions with a bridging oligonucleotide to produce a ligated interaction determining oligonucleotide, wherein the bridging oligonucleotide comprises two hybridization regions capable of specifically binding to the bridging oligonucleotide hybridization regions of the first pair of interaction determining compositions; A method for generating a plurality of barcoded interaction-determining oligonucleotides by barcoding the ligated interaction-determining oligonucleotides using a plurality of barcodes, comprising: each of the plurality of barcodes comprising a barcode sequence and a capture sequence; Obtaining sequencing data of the plurality of barcoded interaction-determining oligonucleotides; determining the interaction between the first and second protein targets based on the association of the interaction determining sequences of the first pair of interaction determining compositions in the obtained sequencing data.

375. 1. A method for determining interactions between cellular component targets, comprising: contacting the cell with a first pair of interaction determining compositions, the cell comprises a first cellular component target and a second cellular component target; each of the first pair of interaction-determining compositions comprises a cellular component binding reagent associated with an interaction-determining oligonucleotide, wherein the cellular component binding reagent of one of the first pair of interaction-determining compositions is capable of specifically binding to the first cellular component target, and the cellular component binding reagent of the other of the first pair of interaction-determining compositions is capable of specifically binding to the second cellular component target; the interaction determining oligonucleotides comprise an interaction determining sequence and a bridging oligonucleotide hybridization region, and the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; ligating the interaction determining oligonucleotides of the first pair of interaction determining compositions with a bridging oligonucleotide to produce a ligated interaction determining oligonucleotide, wherein the bridging oligonucleotide comprises two hybridization regions capable of specifically binding to the bridging oligonucleotide hybridization regions of the first pair of interaction determining compositions; A method for generating a plurality of barcoded interaction-determining oligonucleotides by barcoding the ligated interaction-determining oligonucleotides using a plurality of barcodes, comprising: each of the plurality of barcodes comprising a barcode sequence and a capture sequence; Obtaining sequencing data of the plurality of barcoded interaction-determining oligonucleotides; determining an interaction between the first and second cellular component targets based on the association of the interaction determining sequences of the first pair of interaction determining compositions in the obtained sequencing data.

376. The method of claim 375, wherein at least one of the two cellular component binding reagents comprises a protein binding reagent, the protein binding reagent is associated with one of the two interaction determining oligonucleotides, and the one or more cellular component targets comprises at least one protein target.

377. contacting the cell with the first pair of interaction determining compositions, 377. The method of any one of claims 374 to 376, comprising contacting said cell sequentially or simultaneously with each of said first pair of interaction determining compositions.

378. 378. The method of any one of claims 374 to 377, wherein the first protein target is the same as the second protein target.

379. 379. The method of any one of claims 374-378, wherein the first protein target is different from the second protein target.

380. 380. The method of any one of claims 374 to 379, wherein the interaction determining sequence is 6 to 60 nucleotides in length.

381. The kit of any one of claims 374 to 380, wherein the interaction-determining oligonucleotide is 50 to 500 nucleotides in length.

382. contacting the cell with a second pair of interaction determining compositions; the cell comprises a third protein target and a fourth protein target; 382. The method of any one of claims 374 to 381, wherein each of the second pair of interaction determining compositions comprises a protein binding reagent associated with an interaction determining oligonucleotide, wherein the protein binding reagent of one of the second pair of interaction determining compositions is capable of specifically binding to the third protein target, and the protein binding reagent of the other of the second pair of interaction determining compositions is capable of specifically binding to the fourth protein target.

383. 383. The method of claim 382, ​​wherein at least one of the third and fourth protein targets is different from one of the first and second protein targets.

384. 383. The method of claim 382, ​​wherein at least one of the third and fourth protein targets and at least one of the first and second protein targets are identical.

385. 385. The method of any one of claims 374 to 384, comprising contacting the cell with three or more pairs of interaction determining compositions.

386. 386. The method of claim 385, wherein the interaction determining sequences of at least 10, 100, 1000, or any combination thereof, interaction determining compositions of said plurality of pairs of interaction determining compositions comprise different sequences.

387. 387. The method of any one of claims 374 to 386, wherein the bridging oligonucleotide hybridization regions of the first pair of interaction determining compositions comprise different sequences.

388. 388. The method of any one of claims 374 to 387, wherein at least one of the bridging oligonucleotide hybridization regions is complementary to at least one of the two hybridization regions of the bridging oligonucleotide.

389. ligating the interaction-determining oligonucleotides of the first pair of interaction-determining oligonucleotides of the interaction-determining composition with the bridging oligonucleotide, hybridizing a first hybridization region of the bridging oligonucleotide with a first bridging oligonucleotide hybridization region of the bridging oligonucleotide hybridization region of the interaction-determining oligonucleotide; hybridizing a second hybridization region of the bridging oligonucleotide with a second bridging oligonucleotide hybridization region of the bridging oligonucleotide hybridization region of the interaction-determining oligonucleotide; The method of any one of claims 374 to 388, comprising a step of ligating the interaction determining oligonucleotide hybridized to the bridging oligonucleotide to produce a ligated interaction determining oligonucleotide.

390. 390. The method of any one of claims 374 to 389, wherein the protein binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, an integrin, or a combination thereof.

391. 391. The method of any one of claims 374 to 390, wherein the interaction-determining oligonucleotide is attached to the protein-binding reagent via a linker.

392. 392. The method of any one of claims 374-391, wherein said at least one of said one or more protein targets is on a cell surface.

393. 393. The method of any one of claims 374 to 392, comprising fixing the cells prior to contacting the cells with the first pair of interaction determining compositions.

394. 394. The method of any one of claims 374 to 393, comprising the step of removing unbound interaction determining compositions of said first pair of interaction determining compositions.

395. 395. The method of claim 394, wherein the step of removing the unbound interaction determining composition comprises washing the cells with a wash buffer.

396. 395. The method of claim 394, wherein the step of removing the unbound interaction determining composition comprises selecting the cells using flow cytometry.

397. 397. The method of any one of claims 374 to 396, comprising lysing the cells.

398. The method of any one of claims 374 to 397, wherein the interaction-determining oligonucleotide is configured to be detachable or non-detachable from the protein-binding reagent.

399. The method of any one of claims 374 to 398, comprising a step of cleaving the interaction-determining oligonucleotide from the protein-binding reagent.

400. The method of claim 399, wherein the step of cleaving the interaction-determining oligonucleotide comprises cleaving the interaction-determining oligonucleotide from the protein-binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof.

401. The method of any one of claims 374 to 400, wherein the interaction determining oligonucleotide is not homologous to the genomic sequence of the cell, or the interaction determining oligonucleotide is homologous to the genomic sequence of one species.

402. 402. The method of claim 401, wherein the species is a non-mammalian species.

403. The method of any one of claims 374 to 402, wherein the cell is a tumor cell or a non-tumor cell.

404. 404. The method of any one of claims 374-403, wherein the cell comprises a mammalian cell, a bacterial cell, a viral cell, a yeast cell, a fungal cell, or any combination thereof.

405. 405. The method of any one of claims 374-404, comprising contacting two or more cells with said first pair of interaction determining compositions, wherein each of said two or more cells comprises said first and second protein targets.

406. 406. The method of claim 405, wherein at least one of said two or more cells comprises a single cell.

407. The method of any one of claims 374-406, wherein the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

408. The method of claim 407, wherein at least two barcodes of the plurality of barcodes comprise the same cell marker sequence.

409. The method of any one of claims 374 to 408, wherein the interaction-determining oligonucleotide of one of the first pair of interaction-determining compositions comprises a sequence complementary to the capture sequence.

410. 410. The method of claim 409, wherein the capture sequence comprises a poly(dT) region.

411. The method of any one of claims 409 to 410, wherein the sequence of the interaction-determining oligonucleotide complementary to the capture sequence comprises a poly(dA) region.

412. The method of any one of claims 374 to 411, wherein the interaction-determining oligonucleotide comprises a second barcode sequence.

413. The method of any one of claims 374 to 412, wherein the other of the first pair of interaction-determining oligonucleotides of the interaction identification composition comprises a binding site for a universal primer.

414. 414. The method of any one of claims 374-413, wherein the protein target comprises an extracellular protein, an intracellular protein, or any combination thereof.

415. 415. The method of claim 414, wherein the protein target comprises a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an integrin, or any combination thereof.

416. 416. The method of any one of claims 374-415, wherein the protein target comprises a lipid, a carbohydrate, or any combination thereof.

417. 417. The method of any one of claims 374 to 416, wherein the protein target is selected from a group comprising 10 to 100 different protein targets.

418. 418. The method of any one of claims 374 to 417, wherein the protein-binding reagent is associated with two or more interaction-determining oligonucleotides having the same sequence.

419. 419. The method of any one of claims 374 to 418, wherein the protein binding reagent is associated with two or more interaction-determining oligonucleotides having different interaction-determining sequences.

420. 420. The method of any one of claims 374 to 419, wherein one of said plurality of interaction determining compositions comprises a second protein binding reagent unassociated with said interaction determining oligonucleotide.

421. 421. The method of claim 420, wherein the protein binding reagent and the second protein binding reagent are the same.

422. 422. The method of any one of claims 374 to 421, wherein the protein-binding reagent is associated with a detectable moiety.

423. The method of any one of claims 374 to 422, wherein the plurality of barcodes are associated with particles.

424. 424. The method of claim 423, wherein at least one barcode of the plurality of barcodes is immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or a combination thereof.

425. 425. The method of any one of claims 423 to 424, wherein the particles are disintegrating.

426. 426. The method of any one of claims 423 to 425, wherein the particles comprise beads.

427. 427. The method of any one of claims 423-426, wherein the particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; or wherein the particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogels, paramagnetic materials, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

428. The method of any one of claims 423 to 427, wherein the particles comprise collapsible hydrogel particles.

429. 429. The method of any one of claims 423 to 428, wherein the particle is associated with a detectable moiety.

430. 430. The method of any one of claims 423 to 429, wherein the interaction-determining oligonucleotide is associated with a detectable moiety.

431. 431. The method of any one of claims 423-430, wherein the barcode of the particle comprises a barcode sequence selected from at least 1,000, 10,000, or a combination thereof, different barcode sequences.

432. The method of any one of claims 374 to 431, wherein the barcode sequence of the barcode comprises a random sequence.

433. The method of any one of claims 423 to 432, wherein the particles comprise at least 10,000 barcodes.

434. barcoding the interaction-determining oligonucleotides using the plurality of barcodes, contacting the plurality of barcodes with the interaction determining oligonucleotide to generate barcodes hybridized to the interaction determining oligonucleotide; and extending the barcode hybridized to the interaction-determining oligonucleotide to generate the plurality of barcoded interaction-determining oligonucleotides.

435. 435. The method of claim 434, wherein extending the barcode comprises using a DNA polymerase to extend the barcode to generate the plurality of barcoded interaction-determining oligonucleotides.

436. 435. The method of claim 434, wherein extending the barcodes comprises using a reverse transcriptase to extend the barcodes to generate the plurality of barcoded interaction-determining oligonucleotides.

437. 435. The method of claim 434, wherein extending the barcodes comprises using Moloney murine leukemia virus (M-MLV) reverse transcriptase or Taq DNA polymerase to extend the barcodes to generate the plurality of barcoded interaction-determining oligonucleotides.

438. The method of any one of claims 434 to 437, wherein the step of extending the barcode comprises a step of displacing the bridging oligonucleotide from the ligated interaction-determining oligonucleotide.

439. The method of any one of claims 434 to 438, comprising amplifying said plurality of barcoded interaction-determining oligonucleotides to generate a plurality of amplicons.

440. The method of claim 439, wherein the step of amplifying the plurality of barcoded interaction determining oligonucleotides comprises a step of amplifying at least a portion of the barcode sequence and at least a portion of the interaction determining oligonucleotides using polymerase chain reaction (PCR).

441. The method of any one of claims 439 to 440, wherein the step of obtaining the sequencing data of the plurality of barcoded interaction-determining oligonucleotides comprises the step of obtaining sequencing data of the plurality of amplicons.

442. The method of claim 441, wherein the step of obtaining sequencing data comprises sequencing at least a portion of the barcode sequence and at least a portion of the interaction determining oligonucleotide.

443. The method of any one of claims 374 to 442, wherein the step of obtaining sequencing data of the plurality of barcoded interaction determining oligonucleotides comprises obtaining partial and / or complete sequences of the plurality of barcoded interaction determining oligonucleotides.

444. the plurality of barcodes comprises a plurality of probabilistic barcodes; the barcode sequence of each of the plurality of probabilistic barcodes comprises a molecular beacon sequence; the molecular beacon sequences of at least two of the plurality of probabilistic barcodes comprise different sequences; The method of any one of claims 374 to 443, wherein the step of barcoding the interaction-determining oligonucleotides using the plurality of barcodes to generate the plurality of barcoded interaction-determining oligonucleotides comprises the step of stochastically barcoding the interaction-determining oligonucleotides using the plurality of stochastic barcodes to generate a plurality of stochastically barcoded interaction-determining oligonucleotides.

445. barcoding a plurality of targets of the cell using the plurality of barcodes to generate a plurality of barcoded targets; and obtaining sequencing data for the barcoded target.

446. barcoding the plurality of targets using the plurality of barcodes to generate the plurality of barcoded targets; contacting a copy of the target with the target binding region of the barcode; and reverse transcribing the plurality of targets using the plurality of barcodes to generate a plurality of reverse-transcribed targets.

447. 447. The method of any one of claims 445 to 446, comprising amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining the sequencing data for the plurality of barcoded targets.

448. 448. The method of claim 447, wherein amplifying the barcoded target to generate the plurality of amplified barcoded targets comprises amplifying the barcoded target by polymerase chain reaction (PCR).

449. The method of any one of claims 445 to 448, wherein the step of barcoding the plurality of targets of the cells using the plurality of barcodes to generate the plurality of barcoded targets comprises the step of probabilistically barcoding the plurality of targets of the cells using the plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

450. A kit for identifying protein-protein interactions, comprising: a first pair of interaction-determining compositions, each of said first pair of interaction-determining compositions comprises a protein-binding reagent associated with an interaction-determining oligonucleotide; one said protein binding reagent of said first pair of interaction-determining compositions is capable of specifically binding to a first protein target, and the other protein binding reagent of said first pair of interaction-determining compositions is capable of specifically binding to said second protein target; the interaction-determining oligonucleotide comprises an interaction-determining sequence and a bridging oligonucleotide hybridization region; a first pair of interaction determining compositions, wherein the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; and a plurality of bridging oligonucleotides, each comprising two hybridization regions capable of specifically binding to said bridging oligonucleotide hybridization regions of said first pair of said interaction determining composition.

451. A kit for identifying interactions between cellular components, comprising: a first pair of interaction-determining compositions, each of said first pair of interaction-determining compositions comprises a cellular component-binding reagent associated with an interaction-determining oligonucleotide; one cellular component binding reagent of the first pair of interaction-determining compositions is capable of specifically binding to a first cellular component target, and the other cellular component binding reagent of the first pair of interaction-determining compositions is capable of specifically binding to the second cellular component target; the interaction-determining oligonucleotide comprises an interaction-determining sequence and a bridging oligonucleotide hybridization region; a first pair of interaction determining compositions, wherein the interaction determining sequences of the first pair of interaction determining compositions comprise different sequences; and a plurality of bridging oligonucleotides, each comprising two hybridization regions capable of specifically binding to said bridging oligonucleotide hybridization regions of said first pair of said interaction determining composition.

452. The kit of claim 451, wherein the cellular component binding reagent comprises a protein binding reagent, the first cellular component target comprises a first protein target, and the second cellular component target comprises a second protein target.

453. The kit of any one of claims 450 to 452, wherein the interaction determining sequence is 6 to 60 nucleotides in length.

454. The kit of any one of claims 450 to 453, wherein the interaction-determining oligonucleotide is 50 to 500 nucleotides in length.

455. The kit of any one of claims 450 to 454, further comprising a second pair of interaction determining compositions, each of the second pair of interaction determining compositions comprising a cellular component binding reagent associated with an interaction determining oligonucleotide, wherein one of the cellular component binding reagents of the second pair of interaction determining compositions is capable of specifically binding to a third cellular component target, and the other of the cellular component binding reagents of the second pair of interaction determining compositions is capable of specifically binding to a fourth cellular component target.

456. The kit of any one of claims 455, wherein the third cellular component target comprises a third protein target and the fourth cellular component target comprises a fourth protein target.

457. The kit of any one of claims 455 to 456, wherein at least one of the third and fourth cellular component targets is different from one of the first and second cellular component targets.

458. The kit of any one of claims 455 to 456, wherein at least one of the third and fourth cellular component targets and at least one of the first and second cellular component targets are identical.

459. The kit of any one of claims 450 to 458, wherein the kit comprises three or more pairs of interaction-determining compositions.

460. 460. The kit of claim 459, wherein the interaction determining sequences of at least 10, 100, 1000, or any combination thereof, of the three or more pairs of interaction determining compositions comprise different sequences.

461. 461. The kit of any one of claims 450-460, wherein the bridging oligonucleotide hybridization regions of two interaction determining compositions of the plurality of interaction determining compositions comprise different sequences.

462. 462. The kit of any one of claims 450 to 461, wherein at least one of the bridging oligonucleotide hybridization regions is complementary to at least one of the two hybridization regions of the bridging oligonucleotide.

463. 463. The kit of any one of claims 450-462, wherein the protein-binding reagent or the cellular component-binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof.

464. The kit of any one of claims 450 to 463, wherein the interaction-determining oligonucleotide is bound to the protein-binding reagent or the cellular component-binding reagent via a linker.

465. The kit of any one of claims 450 to 464, wherein the interaction-determining oligonucleotide is not homologous to a genomic sequence of any cell of the subject.

466. The kit of claim 465, wherein the subject's cells are tumor cells or non-tumor cells.

467. 467. The kit of any one of claims 465-466, wherein the cell of interest is a single cell, a mammalian cell, a bacterial cell, a viral cell, a yeast cell, a fungal cell, or any combination thereof.

468. The kit of any one of claims 450-467, further comprising a plurality of barcodes, each of the plurality of barcodes comprising a barcode sequence and a capture sequence.

469. The kit of claim 468, wherein one of the interaction determining oligonucleotides of the first pair of interaction determining compositions comprises a sequence complementary to the capture sequence of at least one barcode of a plurality of barcodes.

470. The kit of any one of claims 468 to 469, wherein the capture sequence comprises a poly(dT) region.

471. The kit of any one of claims 469 to 470, wherein the sequence of the interaction-determining oligonucleotide complementary to the capture sequence of the barcode comprises a poly(dA) region.

472. The kit of any one of claims 468 to 471, wherein the other interaction-determining oligonucleotide of the first pair of interaction identification compositions comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

473. the plurality of barcodes comprises a plurality of probabilistic barcodes; the barcode sequence of each of the plurality of probabilistic barcodes comprises a molecular beacon sequence; The kit of any one of claims 468 to 472, wherein the molecular marker sequences of at least two probabilistic barcodes of the plurality of probabilistic barcodes comprise different sequences.

474. 474. The kit of any one of claims 450-473, wherein the protein target or the cellular component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

475. The kit of any one of claims 450 to 474, wherein the protein target is selected from a group comprising 10 to 100 different protein targets, or the cellular component target is selected from a group comprising 10 to 100 different cellular component targets.

476. 476. The kit of any one of claims 450 to 475, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more interaction-determining oligonucleotides having the same sequence.

477. 478. The kit of any one of claims 450 to 477, wherein the protein-binding reagent or the cellular component-binding reagent is associated with two or more interaction-determining oligonucleotides having different interaction-determining sequences.

478. The kit of any one of claims 450 to 477, wherein one of the first pair of interaction determining compositions comprises a second protein binding reagent or a second cellular component binding reagent that is not associated with the interaction determining oligonucleotide.

479. The kit of claim 478, wherein the first protein binding reagent and the second protein binding reagent are the same, or the first cellular component binding reagent and the second cellular component binding reagent are the same.

480. 480. The kit of any one of claims 450 to 479, wherein the protein-binding reagent or the cellular component-binding reagent is associated with a detectable moiety.

481. The kit of any one of claims 450 to 480, wherein the plurality of barcodes are associated with particles.

482. The kit of claim 481, wherein at least one barcode of the plurality of barcodes is fixed on the particle, partially fixed on the particle, encapsulated in the particle, partially encapsulated in the particle, or a combination thereof.

483. The kit of any one of claims 481 to 482, wherein the particles are disintegrating.

484. The kit of any one of claims 481-483, wherein the particles comprise beads.

485. 485. The kit of any one of claims 481-484, wherein the particles comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; or wherein the particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogels, paramagnetic materials, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

486. The kit of any one of claims 481 to 485, wherein the particles comprise collapsible hydrogel particles.

487. The kit of any one of claims 481 to 486, wherein the particle is associated with a detectable moiety.

488. The kit of any one of claims 481 to 487, wherein the interaction-determining oligonucleotide is associated with a detectable moiety.

489. The kit of any one of claims 481-488, wherein the barcodes of the particles comprise a barcode sequence selected from at least 1000, 1000, or a combination thereof, different barcode sequences.

490. The kit of any one of claims 481-489, wherein the barcode sequence of the barcode comprises a random sequence.

491. The kit of any one of claims 481 to 490, wherein the particles comprise at least 10,000 barcodes.

492. 492. The kit of any one of claims 450 to 491, comprising a DNA polymerase, a reverse transcriptase, or a fixative, or a combination thereof.

493. 493. The kit of any one of claims 450 to 492, comprising Moloney murine leukemia virus (M-MLV) reverse transcriptase or Taq DNA polymerase.

494. 1. A method for sample identification, comprising: contacting one or more cells from each of a plurality of samples with one sample-indexing composition of a plurality of sample-indexing compositions; the one or more cells comprise one or more cellular component targets, each of the plurality of sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotides comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; amplifying the plurality of barcoded, sample-indexed oligonucleotides with a plurality of daisy-chained amplification primers to generate a plurality of daisy-chained extension amplicons; obtaining sequencing data of the plurality of daisy-chained extension amplicons, the sequencing data comprising the plurality of barcoded, sample-indexed oligonucleotides; and identifying a sample origin of at least one of the one or more cells based on the sample index sequence of at least one barcoded sample index oligonucleotide of the plurality of barcoded sample index oligonucleotides.

495. 495. The method of claim 494, wherein the sample index addition sequence is 6 to 60 nucleotides in length.

496. 496. The method of any one of claims 494-495, wherein the sample-indexing oligonucleotide is 50 to 500 nucleotides in length.

497. 497. The method of any one of claims 494 to 496, wherein the sample indexing sequences of at least 10, 100, 1000, or a combination thereof, of the plurality of sample indexing compositions comprise different sequences.

498. 498. The method of any one of claims 494 to 497, wherein the cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof.

499. 500. The method of any one of claims 494 to 498, comprising removing unbound sample indexing compositions of said plurality of sample indexing compositions.

500. 500. The method of any one of claims 494 to 499, comprising lysing the one or more cells from each of the plurality of samples.

501. The method of any one of claims 494 to 500, wherein the sample-indexing oligonucleotide is configured to be detachable or non-detachable from the cellular component binding reagent.

502. 502. The method of any one of claims 494 to 501, comprising the step of cleaving the sample-indexing oligonucleotide from the cellular component binding reagent.

503. The method of claim 502, wherein the step of cleaving the sample index-added oligonucleotide comprises cleaving the sample index-added oligonucleotide from the cellular component binding reagent by UV light cleavage, chemical treatment, heating, enzymatic treatment, or any combination thereof.

504. 504. The method of any one of claims 494-503, wherein said sample-indexing oligonucleotide is not homologous to any genomic sequence of said one or more cells.

505. 504. The method of any one of claims 494-503, wherein the sample-indexing oligonucleotide is homologous to a genomic sequence of a species.

506. 506. The method of any one of claims 494-505, wherein the sample-indexing oligonucleotide comprises a sequence complementary to a capture sequence of at least one barcode of the plurality of barcodes.

507. 507. The method of claim 506, wherein the barcode comprises a target binding region comprising the capture sequence.

508. The method of claim 507, wherein the target binding region comprises a poly(dT) region.

509. 509. The method of any one of claims 506 to 508, wherein the sequence of the sample-indexing oligonucleotide complementary to the capture sequence of the barcode comprises a poly(dA) region.

510. 510. The method of any one of claims 494 to 509, wherein the sample-indexing oligonucleotide comprises a binding site for a molecular beacon sequence, universal.

511. 511. The method of any one of claims 494-510, wherein the cellular component target comprises an extracellular protein, an intracellular protein, or any combination thereof.

512. 512. The method of any one of claims 494-511, wherein the cellular component binding reagent is associated with two or more sample-indexing oligonucleotides having the same sequence.

513. 512. The method of any one of claims 494 to 511, wherein the cellular component binding reagent is associated with two or more sample indexing oligonucleotides having different sample indexing sequences.

514. a barcode of the plurality of barcodes comprising a target binding region and a molecular beacon sequence; The method of any one of claims 494 to 513, wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different molecular beacon sequences.

515. 515. The method of claim 514, wherein the barcode comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof.

516. 516. The method of any one of claims 514-515, wherein the target binding region comprises a poly(dT) region.

517. 517. The method of any one of claims 514 to 516, wherein the plurality of barcodes are associated with particles.

518. The method of claim 517, wherein the particles comprise collapsible hydrogel beads.

519. 519. The method of any one of claims 517-518, wherein the barcode of the particle comprises a molecular beacon sequence selected from at least 1000 different molecular beacon sequences.

520. 520. The method of claim 519, wherein the molecular marker sequence of the barcode comprises a random sequence.

521. 521. The method of any one of claims 517 to 520, wherein the particles comprise at least 10,000 barcodes.

522. barcoding said sample-indexed oligonucleotides using said plurality of barcodes, contacting the plurality of barcodes with the sample-indexed oligonucleotides to produce barcodes hybridized to the sample-indexed oligonucleotides; and extending the barcode hybridized to the sample-indexed oligonucleotide to generate the plurality of barcoded sample-indexed oligonucleotides.

523. 523. The method of claim 522, wherein extending the barcodes comprises using a DNA polymerase to extend the barcodes to generate the plurality of barcoded sample-indexed oligonucleotides.

524. 523. The method of claim 522, wherein extending the barcodes comprises using a reverse transcriptase to extend the barcodes to generate the plurality of barcoded sample-indexed oligonucleotides.

525. 525. The method of any one of claims 494-524, wherein amplifying the plurality of barcoded, sample-indexed oligonucleotides comprises amplifying the plurality of barcoded, sample-indexed oligonucleotides using polymerase chain reaction (PCR).

526. 526. The method of any one of claims 494-525, wherein amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

527. 527. The method of any one of claims 494 to 526, wherein each daisy-chained amplification primer of the plurality of daisy-chained amplification primers comprises a barcoded sample indexing oligonucleotide binding region and an overhang region, wherein the barcoded sample indexing oligonucleotide binding region is capable of binding to the daisy-chained sample indexing region of the sample indexing oligonucleotide.

528. 528. The method of claim 527, wherein said barcoded sample indexing oligonucleotide binding region is between 20 and 200 nucleotides in length.

529. 529. The method of any one of claims 527-528, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

530. 530. The method of any one of claims 527-529, wherein the plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

531. 531. The method of any one of claims 527-530, wherein the overhang region is 50-500 nucleotides in length.

532. 532. The method of any one of claims 527-531, wherein the overhang region comprises a daisy-chained amplification primer barcode sequence.

533. 533. The method of any one of claims 532, wherein two daisy-chained amplification primers of said plurality of daisy-chained amplification primers comprise overhang regions having identical daisy-chained amplification primer barcode sequences.

534. 533. The method of any one of claims 532, wherein two daisy-chained amplification primers of said plurality of daisy-chained amplification primers comprise an overhang region having two daisy-chained amplification primer barcode sequences.

535. 533. The method of any one of claims 532, wherein the overhang regions of the plurality of daisy-chained amplification primers comprise different daisy-chained amplification primer barcode sequences.

536. 536. The method of any one of claims 527-535, wherein a daisy-chained extension amplicon of said plurality of daisy-chained extension amplicons is between 250 and 1000 nucleotides in length.

537. 537. The method of any one of claims 494-536, wherein obtaining the sequencing data comprises sequencing at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotide.

538. 538. The method of any one of claims 494 to 537, wherein identifying the sample origin of the at least one cell comprises identifying the sample origin of the plurality of barcoded targets based on the sample indexing sequence of the at least one barcoded sample indexing oligonucleotide.

539. The method of any one of claims 494 to 538, wherein barcoding the sample-indexed oligonucleotides using the plurality of barcodes to generate the plurality of barcoded sample-indexed oligonucleotides comprises probabilistically barcoding the sample-indexed oligonucleotides using a plurality of probabilistic barcodes to generate a plurality of probabilistically barcoded sample-indexed oligonucleotides.

540. barcoding a plurality of targets of the cells using the plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a cell-labeling sequence, and at least two barcodes of the plurality of barcodes comprise the same cell-labeling sequence; and obtaining sequencing data for the barcoded target.

541. barcoding the plurality of targets using the plurality of barcodes to generate the plurality of barcoded targets; contacting a copy of the target with the target binding region of the barcode; and reverse transcribing said plurality of targets using said plurality of barcodes to generate a plurality of reverse transcribed targets.

542. 542. The method of any one of claims 540 to 541, comprising amplifying the barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining the sequencing data for the plurality of barcoded targets.

543. 543. The method of claim 542, wherein amplifying the barcoded target to generate the plurality of amplified barcoded targets comprises amplifying the barcoded target by polymerase chain reaction (PCR).

544. The method of any one of claims 540 to 543, wherein barcoding the plurality of targets of the cells using the plurality of barcodes to generate the plurality of barcoded targets comprises probabilistically barcoding the plurality of targets of the cells using a plurality of probabilistic barcodes to generate a plurality of probabilistic barcoded targets.

545. 1. A method for sample identification, comprising: contacting one or more cells from each of a plurality of samples with one sample-indexing composition of a plurality of sample-indexing compositions; each of the one or more cells comprises one or more binding targets; each of the plurality of sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more binding targets; the sample indexing oligonucleotides comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; barcoding the sample-indexed oligonucleotides with a plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides using a plurality of daisy-chained amplification primers; and identifying a sample origin of at least one of the one or more cells based on the sample index sequence of at least one barcoded sample index oligonucleotide of the plurality of barcoded sample index oligonucleotides.

546. barcoding said sample-indexed oligonucleotides using said plurality of barcodes, contacting the plurality of barcodes with the sample-indexed oligonucleotides to produce barcodes hybridized to the sample-indexed oligonucleotides; and extending the barcode hybridized to the sample index-added oligonucleotide to generate the plurality of barcoded sample index-added oligonucleotides.

547. 547. The method of claim 546, wherein extending the barcode comprises using a DNA polymerase to extend the barcode to generate the plurality of barcoded sample-indexed oligonucleotides.

548. 547. The method of claim 546, wherein extending the barcode comprises using a reverse transcriptase to extend the barcode to generate the plurality of barcoded sample-indexed oligonucleotides.

549. 549. The method of any one of claims 546-548, comprising amplifying said plurality of barcoded sample-indexed oligonucleotides.

550. 550. The method of claim 549, wherein amplifying the plurality of barcoded sample index-added oligonucleotides comprises amplifying the plurality of barcoded sample index-added oligonucleotides using polymerase chain reaction (PCR).

551. 551. The method of any one of claims 549-550, wherein amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

552. 552. The method of any one of claims 549-551, wherein amplifying said plurality of barcoded sample-indexed oligonucleotides comprises amplifying said plurality of barcoded sample-indexed oligonucleotides with said plurality of daisy-chained amplification primers to generate said plurality of daisy-chained extension amplicons.

553. The method of claim 552, wherein the daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise a barcoded sample index-added oligonucleotide binding region and an overhang region, and the barcoded sample index-added oligonucleotide binding region is capable of binding to the daisy-chained sample index-added region of the sample index-added oligonucleotide.

554. 554. The method of claim 553, wherein said barcoded sample indexing oligonucleotide binding region is between 20 and 200 nucleotides in length.

555. 555. The method of any one of claims 553-554, wherein two daisy-chained amplification primers of said plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

556. 555. The method of any one of claims 553-554, wherein the plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

557. 557. The method of any one of claims 553-556, wherein the overhang region is 50 to 500 nucleotides in length.

558. 558. The method of any one of claims 553-557, wherein the overhang region comprises a daisy-chained amplification primer barcode sequence.

559. The method of any one of claims 558, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise overhang regions having identical daisy-chained amplification primer barcode sequences.

560. The method of any one of claims 558, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise an overhang region having two daisy-chained amplification primer barcode sequences.

561. The method of any one of claims 558, wherein the overhang regions of the multiple daisy-chained amplification primers comprise different daisy-chained amplification primer barcode sequences.

562. 562. The method of any one of claims 553-561, wherein a daisy-chained extension amplicon of said plurality of daisy-chained extension amplicons is between 250 and 1000 nucleotides in length.

563. 563. The method of any one of claims 545-562, wherein acquiring said sequencing data for said plurality of barcoded sample-indexed oligonucleotides comprises acquiring sequencing data for said plurality of daisy-chained extension amplicons.

564. 1. A method for sample identification, comprising: contacting one or more cells from each of a plurality of samples with one sample-indexing composition of a plurality of sample-indexing compositions; each of the one or more cells comprises one or more cellular component targets; each of the plurality of sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide, the cellular component binding reagent capable of specifically binding to at least one of the one or more cellular component targets; the sample indexing oligonucleotides comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions comprise different sequences; and identifying the sample origin of at least one of the one or more cells based on the sample index sequence of at least one sample index oligonucleotide of the plurality of sample index compositions and a plurality of daisy-chained amplification primers.

565. identifying the sample origin of the at least one cell, barcoding the sample-indexed oligonucleotides of said plurality of sample-indexed compositions with the plurality of barcodes to generate a plurality of barcoded sample-indexed oligonucleotides; obtaining sequencing data for the plurality of barcoded, sample-indexed oligonucleotides; and identifying the sample origin of the cell based on the sample index sequence of at least one barcoded sample index oligonucleotide of the plurality of barcoded sample index oligonucleotides in the sequencing data.

566. barcoding said sample-indexed oligonucleotides using said plurality of barcodes, contacting the plurality of barcodes with the sample-indexed oligonucleotides to produce barcodes hybridized to the sample-indexed oligonucleotides; and extending the barcodes hybridized to the sample index-added oligonucleotides to generate the plurality of barcoded sample index-added oligonucleotides.

567. 567. The method of claim 566, wherein extending the barcode comprises using a DNA polymerase to extend the barcode to generate the plurality of barcoded sample-indexed oligonucleotides.

568. 567. The method of claim 566, wherein extending the barcode comprises using a reverse transcriptase to extend the barcode to generate the plurality of barcoded sample-indexed oligonucleotides.

569. 569. The method of any one of claims 565-568, comprising amplifying said plurality of barcoded sample-indexed oligonucleotides.

570. 570. The method of claim 569, wherein amplifying the plurality of barcoded sample index-added oligonucleotides comprises amplifying the plurality of barcoded sample index-added oligonucleotides using polymerase chain reaction (PCR).

571. 571. The method of any one of claims 569-570, wherein amplifying the plurality of barcoded sample-indexing oligonucleotides comprises amplifying at least a portion of the molecular beacon sequence and at least a portion of the sample-indexing oligonucleotides.

572. 572. The method of any one of claims 569-571, wherein amplifying said plurality of barcoded, sample-indexed oligonucleotides comprises amplifying said plurality of barcoded, sample-indexed oligonucleotides with said plurality of daisy-chained amplification primers to generate a plurality of daisy-chained extension amplicons.

573. 573. The method of claim 572, wherein the daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise a barcoded sample index-added oligonucleotide binding region and an overhang region, and the barcoded sample index-added oligonucleotide binding region is capable of binding to the daisy-chained sample index-added region of the sample index-added oligonucleotide.

574. 574. The method of claim 573, wherein said barcoded sample indexing oligonucleotide binding region is 20 to 200 nucleotides in length.

575. 575. The method of any one of claims 573-574, wherein two daisy-chained amplification primers of said plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

576. 575. The method of any one of claims 573-574, wherein the plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

577. 577. The method of any one of claims 573-576, wherein the overhang region is 50 to 500 nucleotides in length.

578. 578. The method of any one of claims 573-577, wherein the overhang region comprises a daisy-chained amplification primer barcode sequence.

579. The method of any one of claims 578, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise overhang regions having identical daisy-chained amplification primer barcode sequences.

580. The method of any one of claims 578, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise an overhang region having two daisy-chained amplification primer barcode sequences.

581. The method of any one of claims 578, wherein the overhang regions of the multiple daisy-chained amplification primers comprise different daisy-chained amplification primer barcode sequences.

582. 582. The method of any one of claims 573-581, wherein the daisy-chained extension amplicons of said plurality of daisy-chained extension amplicons are between 250 and 1000 nucleotides in length.

583. A plurality of sample-indexed compositions, each of said plurality of sample-indexing compositions comprises a cellular component binding reagent associated with a sample-indexing oligonucleotide; the cellular component binding reagent is capable of specifically binding to at least one cellular component target; the sample indexing oligonucleotide comprises a sample indexing sequence for identifying the sample origin of one or more cells of the sample and a daisy-chained amplification primer binding sequence; a plurality of sample indexing compositions, wherein the sample indexing sequences of at least two sample indexing compositions of said plurality of sample indexing compositions comprise different sequences; and a plurality of daisy-chained amplification primers.

584. The kit of claim 583, wherein the daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise a barcoded sample index-added oligonucleotide binding region and an overhang region, and the barcoded sample index-added oligonucleotide binding region is capable of binding to the daisy-chained sample index-added region of the sample index-added oligonucleotide.

585. 585. The kit of claim 584, wherein the barcoded sample indexing oligonucleotide binding region is 20 to 200 nucleotides in length.

586. 586. The kit of any one of claims 584-585, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having identical sequences.

587. 586. The kit of any one of claims 584-585, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise barcoded, sample-indexed oligonucleotide binding regions having different sequences.

588. 588. The kit of any one of claims 586-587, wherein the barcoded sample indexed oligonucleotide binding region comprises the daisy-chained amplification primer binding sequence, its complement, its reverse complement, or a combination thereof.

589. The kit of any one of claims 584 to 588, wherein the daisy-chained amplification primer binding sequences of at least two sample indexing compositions of said plurality of sample indexing compositions comprise identical sequences.

590. The kit of any one of claims 584 to 588, wherein the daisy-chained amplification primer binding sequences of at least two sample indexing compositions of said plurality of sample indexing compositions comprise different sequences.

591. 591. The kit of any one of claims 584-590, wherein the overhang region is 50 to 500 nucleotides in length.

592. 591. The kit of any one of claims 584-590, wherein the overhang region comprises a daisy-chained amplification primer barcode sequence.

593. The kit of any one of claims 592, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise overhang regions having identical daisy-chained amplification primer barcode sequences.

594. The kit of any one of claims 592, wherein two daisy-chained amplification primers of the plurality of daisy-chained amplification primers comprise an overhang region having two daisy-chained amplification primer barcode sequences.

595. The kit of any one of claims 592, wherein the overhang regions of the plurality of daisy-chained amplification primers comprise different daisy-chained amplification primer barcode sequences.

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