Determining 5' transcript sequences

Abstract

To provide a kit for labeling a nucleic acid target in a sample.SOLUTION: A kit comprises: a plurality of oligonucleotide barcodes, in which each of the plurality of oligonucleotide barcodes comprises a molecular label and a target-binding region, and at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences; a reverse transcriptase; a template switching oligonucleotide comprising the target-binding region, or a portion thereof; and a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.SELECTED DRAWING: Figure 4A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 739,795, filed October 1, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes. Reference to sequence listing This application is filed together with an electronic Sequence Listing, which is 4.0 kilobytes in size and has been submitted as file entitled 68EB_298703_WO, created on September 30, 2019. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. The present disclosure relates generally to the field of molecular biology, and more particularly to multi-omics analysis using molecular barcoding. [Background technology]

[0002] Molecular barcoding methods and techniques are useful for single-cell transcriptomics analysis, such as deciphering gene expression profiles to determine the state of a cell using, for example, reverse transcription, polymerase chain reaction (PCR) amplification, and next-generation sequencing (NGS). Molecular barcoding is also useful for single-cell proteomics analysis. There is a need for methods and techniques for molecular barcoding nucleic acid targets at one or both of the 5' and 3' ends. Summary of the Invention

[0003] The present disclosure includes methods for labeling nucleic acid targets in a sample. In some embodiments, the method includes contacting a copy of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copy of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each comprising a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; hybridizing the complement of the target binding region of each barcoded nucleic acid molecule to (i) an oligonucleotide barcode of the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules; and extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each comprising the first molecular label and the second molecular label. The method may include determining the copy number of a nucleic acid target in a sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with a plurality of extended barcoded nucleic acid molecules, or products thereof.

[0004] The disclosure herein includes a method for determining the number of nucleic acid targets in a sample. In some embodiments, the method includes contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; and (i) combining the complement of the target binding region of each barcoded nucleic acid molecule with a plurality of oligonucleotide barcodes. the method includes hybridizing (i) an oligonucleotide barcode among the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules; extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a first molecular label and a second molecular label; and determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with the plurality of extended barcoded nucleic acid molecules, or products thereof.

[0005] The method may include amplifying a plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules, each comprising a first molecular label or a second molecular label, wherein determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels having distinct sequences associated with the plurality of single-labeled nucleic acid molecules. In some embodiments, determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels having distinct sequences associated with the plurality of single-labeled nucleic acid molecules. The method may include amplifying a plurality of extended barcoded nucleic acid molecules to produce a plurality of copies of the extended barcoded nucleic acid molecules, wherein determining the copy number of the nucleic acid target in the sample includes determining the copy number of the nucleic acid target in the sample based on (i) the number of first molecular labels having distinct sequences associated with the plurality of copies of the extended barcoded nucleic acid molecules, or products thereof, and / or (ii) the number of second molecular labels having distinct sequences associated with the plurality of copies of the extended barcoded nucleic acid molecules, or products thereof.

[0006] The disclosure herein includes a method for determining the number of nucleic acid targets in a sample. In some embodiments, the method includes the steps of: contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; and interfering the complement of the target binding region of each barcoded nucleic acid molecule with (i) the plurality of oligonucleotide barcodes. the method further comprises hybridizing (i) an oligonucleotide barcode among the plurality of barcoded nucleic acid molecules, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules; extending the 3' ends of the plurality of barcoded nucleic acid molecules to produce a plurality of extended barcoded nucleic acid molecules, each of which comprises the first molecular label and the second molecular label; amplifying the plurality of extended barcoded nucleic acid molecules to produce a plurality of single-labeled nucleic acid molecules, each of which comprises the first molecular label or the second molecular label; and determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels having distinct sequences associated with the plurality of single-labeled nucleic acid molecules.

[0007] In some embodiments, the method includes determining the copy number of a nucleic acid target in a sample based on the number of first molecular labels having distinct sequences associated with a plurality of single-labeled nucleic acid molecules. In some embodiments, the method includes denaturing the plurality of barcoded nucleic acid molecules before hybridizing the complement of the target binding region of each barcoded nucleic acid molecule with (i) an oligonucleotide barcode among the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules. In some embodiments, the method includes denaturing the plurality of extended barcoded nucleic acid molecules before amplifying the plurality of extended barcoded nucleic acid molecules. In some embodiments, determining the copy number of the nucleic acid target includes determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of second molecular labels having distinct sequences associated with a single-labeled nucleic acid molecule among the plurality of single-labeled nucleic acid molecules comprising the respective sequences of the plurality of nucleic acid targets. In some embodiments, determining the copy number of the nucleic acid target comprises determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of first molecular labels having distinct sequences associated with single-labeled nucleic acid molecules among a plurality of single-labeled nucleic acid molecules comprising the respective sequences of the plurality of nucleic acid targets. In some embodiments, the respective sequences of the plurality of nucleic acid targets comprise respective subsequences of the plurality of nucleic acid targets. In some embodiments, the sequences of the nucleic acid targets in the plurality of barcoded nucleic acid molecules comprise subsequences of the nucleic acid targets.

[0008] In some embodiments, the first molecular label is hybridized to the second molecular label after extending the 3'-end of a plurality of barcoded nucleic acid molecules. In some embodiments, each extended barcoded nucleic acid molecule comprises a first molecular label, a second molecular label, a target binding region, and a complement of the target binding region. In some embodiments, the complement of the target binding region is complementary to a portion of the target binding region. In some embodiments, the target binding region comprises a gene-specific sequence. In some embodiments, the target binding region comprises a poly(dT) sequence.

[0009] In some embodiments, hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with its own target binding region comprises intramolecular hybridization of the target binding region in the barcoded nucleic acid molecule with the complement of the target binding region, forming a stem-loop. In some embodiments, the second molecular label is a complement of the first molecular label. In some embodiments, hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises intermolecular hybridization of the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes. In some embodiments, the second molecular label is different from the first molecular label, and the second molecular label is not a complement of the first molecular label. In some embodiments, the method includes extending the 3' end of an oligonucleotide barcode hybridized to the complement of the target binding region of the barcoded nucleic acid molecule to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a complement of a first molecular label and a second molecular label. In some embodiments, the sequence of the second molecular label is different from the sequence of the first molecular label, and the second molecular label is not a complement of the first molecular label. In some embodiments, hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules includes intermolecular hybridization of the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules. In some embodiments, the sequence of the second molecular label is different from the sequence of the first molecular label, and the second molecular label is not a complement of the first molecular label.

[0010] In some embodiments, the reverse transcriptase is capable of terminal transferase activity. In some embodiments, the template switch oligonucleotide comprises one or more 3' ribonucleotides, e.g., three 3' ribonucleotides. In some embodiments, the 3' ribonucleotide comprises a guanine. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase, e.g., murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0011] In some embodiments, the sample comprises a single cell. In some embodiments, the sample comprises a plurality of cells, a plurality of single cells, a tissue, a tumor sample, or any combination thereof. In some embodiments, the single cell comprises an immune cell. In some embodiments, the immune cell is a B cell or a T cell. In some embodiments, the single cell comprises a circulating tumor cell. In some embodiments, each oligonucleotide barcode comprises a first universal sequence. In some embodiments, the plurality of extended barcoded nucleic acid molecules comprises the first universal sequence and a complement of the first universal sequence. In some embodiments, amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of copies of the extended barcoded nucleic acid molecule comprises using a primer capable of hybridizing to the first universal sequence, or a complement thereof. In some embodiments, amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules comprises using a primer capable of hybridizing to the first universal sequence, or a complement thereof, and an amplification primer. In some embodiments, the amplification primer is a target-specific primer. In some embodiments, the target-specific primers specifically hybridize to an immune receptor. In some embodiments, the target-specific primers specifically hybridize to a constant region of an immune receptor. In some embodiments, the target-specific primers specifically hybridize to a variable region of an immune receptor. In some embodiments, the target-specific primers specifically hybridize to a diversity region of an immune receptor. In some embodiments, the target-specific primers specifically hybridize to the junction of the variable region and diversity region of an immune receptor. In some embodiments, the immune receptor is a T cell receptor (TCR) and / or a B cell receptor (BCR). In some embodiments, the TCR comprises a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof. In some embodiments, the BCR comprises a BCR heavy chain and / or a BCR light chain.

[0012] In some embodiments, extending the 3' ends of the plurality of barcoded nucleic acid molecules comprises extending the 3' ends of the plurality of barcoded nucleic acid molecules using a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. In some embodiments, the DNA polymerase comprises Klenow fragment. In some embodiments, the method comprises obtaining sequence information of the plurality of extended barcoded nucleic acid molecules or products thereof. In some embodiments, obtaining sequence information comprises attaching sequencing adapters to the plurality of extended barcoded nucleic acid molecules or products thereof. In some embodiments, obtaining sequence information comprises attaching sequencing adapters to the plurality of single-labeled nucleic acid molecules or products thereof. In some embodiments, obtaining sequence information comprises obtaining sequence information of a BCR light chain and a BCR heavy chain of a single cell. In some embodiments, the sequence information of the BCR light chain and the BCR heavy chain includes the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the BCR light chain and / or the BCR heavy chain. In some embodiments, the method includes pairing the BCR light chain and the BCR heavy chain of the single cell based on the obtained sequence information. In some embodiments, the sample includes a plurality of single cells, and the method includes pairing the BCR light chain and the BCR heavy chain of at least 50% of the single cells based on the obtained sequence information. In some embodiments, the step of obtaining sequence information includes obtaining sequence information of the TCR alpha chain and the TCR beta chain of the single cell. In some embodiments, the sequence information of the TCR alpha chain and the TCR beta chain includes the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the TCR alpha chain and / or the TCR beta chain. In some embodiments, the method includes pairing the TCR alpha chain and the TCR beta chain of the single cell based on the obtained sequence information.In some embodiments, the sample comprises a plurality of single cells, and the method comprises pairing the TCR alpha chain and the TCR beta chain of at least 50% of the single cells based on the obtained sequence information. In some embodiments, obtaining sequence information comprises obtaining sequence information of the TCR gamma chain and the TCR delta chain of the single cells. In some embodiments, the sequence information of the TCR gamma chain and the TCR delta chain comprises the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the TCR gamma chain and / or the TCR delta chain. In some embodiments, the method comprises pairing the TCR gamma chain and the TCR delta chain of the single cells based on the obtained sequence information. In some embodiments, the sample comprises a plurality of single cells, and the method comprises pairing the TCR gamma chain and the TCR delta chain of at least 50% of the single cells based on the obtained sequence information.

[0013] In some embodiments, the complement of the target binding region comprises the reverse complement of the target binding region. In some embodiments, the complement of the target binding region comprises the complementary sequence of the target binding region. In some embodiments, the complement of the molecular label comprises the reverse complement of the molecular label. In some embodiments, the complement of the molecular label comprises the complementary sequence of the molecular label. In some embodiments, the plurality of barcoded nucleic acid molecules comprise barcoded deoxyribonucleic acid (DNA) molecules. In some embodiments, the barcoded nucleic acid molecules comprise barcoded ribonucleic acid (RNA) molecules. In some embodiments, the nucleic acid target comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA comprising a poly(A) tail, or any combination thereof. In some embodiments, the mRNA encodes an immune receptor. In some embodiments, the nucleic acid target comprises a cellular component binding reagent. In some embodiments, the nucleic acid molecule is associated with a cellular component binding reagent. In some embodiments, the method comprises dissociating the nucleic acid molecule and the cellular component binding reagent. In some embodiments, at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences. In some embodiments, each molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides.

[0014] In some embodiments, a plurality of oligonucleotide barcodes are associated with a solid support. In some embodiments, a plurality of oligonucleotide barcodes associated with the same solid support each comprise the same sample label. In some embodiments, each sample label of the plurality of oligonucleotide barcodes comprises at least six nucleotides. In some embodiments, a plurality of oligonucleotide barcodes each comprise a cell label. In some embodiments, each cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides. In some embodiments, oligonucleotide barcodes associated with the same solid support comprise the same cell label. In some embodiments, oligonucleotide barcodes associated with different solid supports comprise different cell labels. In some embodiments, a plurality of extended barcoded nucleic acid molecules each comprise a cell label and a complement of the cell label. In some embodiments, the complement of the cell label comprises the reverse complementary sequence of the cell label. In some embodiments, the complement of the cell label comprises the complementary sequence of the cell label. In some embodiments, the method comprises extending a plurality of oligonucleotide barcodes hybridized to copies of a nucleic acid target in the presence of one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof. In some embodiments, the solid support comprises synthetic particles. In some embodiments, the solid support comprises a planar surface.

[0015] In some embodiments, the sample includes a single cell, and the method includes associating synthetic particles comprising a plurality of oligonucleotide barcodes with the single cell in the sample. In some embodiments, the method includes lysing the single cell after associating the synthetic particles with the single cell. In some embodiments, lysing the single cell includes heating the sample, contacting the sample with a surfactant, changing the pH of the sample, or any combination thereof. In some embodiments, the synthetic particles and the single cell are in the same well. In some embodiments, the synthetic particles and the single cell are in the same droplet. In some embodiments, at least one of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially immobilized on the synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is encapsulated within the synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially encapsulated within the synthetic particle. In some embodiments, the synthetic particle is disintegratable. In some embodiments, the synthetic particle includes a bead. In some embodiments, 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. In some embodiments, the synthetic 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, or any combination thereof. In some embodiments, the synthetic particles comprise disintegrable hydrogel particles.In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and / or the support functional group and the linker functional group are associated with each other. In some embodiments, the linker functional group and the support functional group are independently selected from the group consisting of C6, biotin, streptavidin, primary amines, aldehydes, ketones, and any combination thereof.

[0016] The present disclosure includes kits. In some embodiments, the kits include a plurality of oligonucleotide barcodes, each of the plurality of oligonucleotide barcodes comprising a molecular label and a target binding region, and at least 10 of the plurality of oligonucleotide barcodes comprising different molecular label sequences; a reverse transcriptase; a template switching oligonucleotide comprising the target binding region, or a portion thereof; and a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

[0017] In some embodiments, the DNA polymerase comprises a Klenow fragment. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is murine leukemia virus (MLV) reverse transcriptase. In some embodiments, the viral reverse transcriptase is Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the template switch oligonucleotide comprises one or more 3' ribonucleotides. In some embodiments, the template switch oligonucleotide comprises three 3' ribonucleotides. In some embodiments, the 3' ribonucleotide comprises guanine. In some embodiments, the kit comprises one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof.

[0018] In some embodiments, the kit includes a buffer. In some embodiments, the kit includes a cartridge. In some embodiments, the kit includes one or more reagents for a reverse transcription reaction. In some embodiments, the kit includes one or more reagents for an amplification reaction. In some embodiments, the target binding regions include gene-specific sequences, oligo(dT) sequences, random multimers, or any combination thereof. In some embodiments, the oligonucleotide barcodes include identical sample labels and / or identical cell labels. In some embodiments, each sample label and / or cell label of the plurality of oligonucleotide barcodes includes at least six nucleotides. In some embodiments, each molecular label of the plurality of oligonucleotide barcodes includes at least six nucleotides. In some embodiments, at least one of the plurality of oligonucleotide barcodes is immobilized on a synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially immobilized on a synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is encapsulated within a synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially encapsulated within a synthetic particle. In some embodiments, the synthetic particle is disintegratable. In some embodiments, the synthetic particle includes a bead. In some embodiments, 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.In some embodiments, the synthetic 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. In some embodiments, the synthetic particles comprise collapsible hydrogel particles. In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particles comprise a solid support functional group, and / or the support functional group and the linker functional group are associated with each other. In some embodiments, the linker functional group and the support functional group are independently selected from the group consisting of C6, biotin, streptavidin, primary amine, aldehyde, ketone, and any combination thereof. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 illustrates a non-limiting exemplary barcode. [Figure 2] FIG. 1 illustrates a non-limiting exemplary workflow for barcoding and electronic counting. [Figure 3] 1 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of 3′-barcoded targets from multiple targets. [Figure 4A] 1 shows a schematic representation of a non-limiting exemplary method for gene-specifically labeling nucleic acid targets at the 5′ end. [Figure 4B] 1 shows a schematic representation of a non-limiting exemplary method for gene-specifically labeling nucleic acid targets at the 5′ end. [Figure 5A] 1 shows a schematic representation of a non-limiting exemplary method for labeling nucleic acid targets at the 5′ end for whole transcriptome analysis. [Figure 5B] 1 shows a schematic representation of a non-limiting exemplary method for labeling nucleic acid targets at the 5′ end for whole transcriptome analysis. [Figure 6A]1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6B] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6C] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6D] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6E] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6F] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6G] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6H] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6I] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6J]1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 6K] 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. [Figure 7] Non-limiting exemplary schematic diagrams are provided for implementing the V(D)J protocol, the antibody-oligonucleotide (AbO) protocol, and the single-cell mRNA expression profiling protocol (e.g., the BD Rhapsody targeted protocol) as one workflow. [Figure 8A]

[0023] Figure 1 shows non-limiting, exemplary experimental results of capturing and sequencing the 5' T cell receptor (TCR) V(D)J region using the V(D)J protocol. The V(D)J hairpin protocol can involve 3' and 5' amplification performed on the same beads using captured mRNA molecules from a single, resting peripheral blood mononuclear cell (PBMC). The TCR alpha / beta chain pairing efficiency was 37.9%, which is comparable to other platforms such as Clonetech's scTCR profiling kit. [Figure 8B]

[0023] Figure 1 shows non-limiting, exemplary experimental results of capturing and sequencing the 5' T cell receptor (TCR) V(D)J region using the V(D)J protocol. The V(D)J hairpin protocol can involve 3' and 5' amplification performed on the same beads using captured mRNA molecules from a single, resting peripheral blood mononuclear cell (PBMC). The TCR alpha / beta chain pairing efficiency was 37.9%, which is comparable to other platforms such as Clonetech's scTCR profiling kit. [Figure 8C]

[0023] Figure 1 shows non-limiting, exemplary experimental results of capturing and sequencing the 5' T cell receptor (TCR) V(D)J region using the V(D)J protocol. The V(D)J hairpin protocol can involve 3' and 5' amplification performed on the same beads using captured mRNA molecules from a single, resting peripheral blood mononuclear cell (PBMC). The TCR alpha / beta chain pairing efficiency was 37.9%, which is comparable to other platforms such as Clonetech's scTCR profiling kit. [Figure 9A-9B] Non-limiting exemplary plots are shown demonstrating improved 5' V(D)J detection sensitivity using an improved V(D)J protocol. Ethylene glycol was added to aid in the reduction of secondary structure during reverse transcription (RT). Hybridization time, buffer, and template switching (TS) oligo-dT length were modified to improve sensitivity. Four libraries were generated and sequenced together: 5' TCR, 5' BCR, 5' 30-plex immune panel, and 3' immune response panel. A more stringent Ampure cleanup (0.6X) was performed. [Figure 10A] Figure 9 shows non-limiting exemplary experimental results of improving 5' V(D)J detection sensitivity using the improved V(D)J protocol described with respect to Figure 9. Compared to the protocol described with respect to Figure 8, TCR alpha / beta pairing efficiency was improved from 37.9% to 52%. 5' heavy and light chain mRNA molecules were successfully detected in B cells. [Figure 10B] Figure 9 shows non-limiting exemplary experimental results of improving 5' V(D)J detection sensitivity using the improved V(D)J protocol described with respect to Figure 9. Compared to the protocol described with respect to Figure 8, TCR alpha / beta pairing efficiency was improved from 37.9% to 52%. 5' heavy and light chain mRNA molecules were successfully detected in B cells. [Figure 10C]Figure 9 shows non-limiting exemplary experimental results of improving 5' V(D)J detection sensitivity using the improved V(D)J protocol described with respect to Figure 9. Compared to the protocol described with respect to Figure 8, TCR alpha / beta pairing efficiency was improved from 37.9% to 52%. 5' heavy and light chain mRNA molecules were successfully detected in B cells. [Figure 10D] Figure 9 shows non-limiting exemplary experimental results of improving 5' V(D)J detection sensitivity using the improved V(D)J protocol described with respect to Figure 9. Compared to the protocol described with respect to Figure 8, TCR alpha / beta pairing efficiency was improved from 37.9% to 52%. 5' heavy and light chain mRNA molecules were successfully detected in B cells. [Figure 10E] Figure 9 shows non-limiting exemplary experimental results of improving 5' V(D)J detection sensitivity using the improved V(D)J protocol described with respect to Figure 9. Compared to the protocol described with respect to Figure 8, TCR alpha / beta pairing efficiency was improved from 37.9% to 52%. 5' heavy and light chain mRNA molecules were successfully detected in B cells. [Figure 11A] 11A, 11C, 11E) and 5′ amplification (FIGS. 11B, 11D, 11F). [Figure 11B] 11A, 11C, 11E) and 5′ amplification (FIGS. 11B, 11D, 11F). [Figure 11C] 11A, 11C, 11E) and 5′ amplification (FIGS. 11B, 11D, 11F). [Figure 11D] 11A, 11C, 11E) and 5′ amplification (FIGS. 11B, 11D, 11F). [Figure 11E] 11A, 11C, 11E) and 5′ amplification (FIGS. 11B, 11D, 11F). [Figure 11F] 11A, 11C, 11E) and 5′ amplification (FIGS. 11B, 11D, 11F). [Figure 12A] 12A, 12C, 12E) and 5′ amplification (FIGS. 12B, 12D, 12F). [Figure 12B] 12A, 12C, 12E) and 5′ amplification (FIGS. 12B, 12D, 12F). [Figure 12C] 12A, 12C, 12E) and 5′ amplification (FIGS. 12B, 12D, 12F). [Figure 12D] 12A, 12C, 12E) and 5′ amplification (FIGS. 12B, 12D, 12F). [Figure 12E]12A, 12C, 12E) and 5′ amplification (FIGS. 12B, 12D, 12F). [Figure 12F] 12A, 12C, 12E) and 5′ amplification (FIGS. 12B, 12D, 12F). DETAILED DESCRIPTION OF THE INVENTION

[0020] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless context dictates 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 will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and make part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety for relevant art.

[0021] Quantifying a small number of nucleic acids, such as messenger ribonucleotide acid (mRNA) molecules, is clinically important, for example, to determine the genes expressed in cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can be very 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 molecular copies in each cycle. However, PCR can have drawbacks because each molecule replicates with a stochastic probability, and this probability varies depending on the PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Stochastic barcodes with unique molecular labels (also called molecular indexes (MIs)) can be used to count the number of molecules and correct for amplification bias. Stochastic barcoding, such as the Precise™ assay (Cellular Research, Inc., Palo Alto, CA) and the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ), can correct for biases induced by the library preparation step by using molecular beacons (MLs) to label mRNA during PCR and reverse transcription (RT).

[0022] The Precise™ assay utilizes a non-depleting pool of stochastic barcodes with a large number of unique molecular tag sequences, e.g., 6561-65536, on poly(T) oligonucleotides to hybridize to 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 hybridizes to a stochastic barcode, resulting in the generation of a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules from each microwell of a microwell plate may be pooled into a single tube for PCR amplification and sequencing. Raw sequencing data can be analyzed to obtain the number of reads, the number of stochastic barcodes with unique molecular tag sequences, and the number of mRNA molecules.

[0023] The present disclosure includes methods for labeling nucleic acid targets in a sample. In some embodiments, the method includes contacting a copy of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copy of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each comprising a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; hybridizing the complement of the target binding region of each barcoded nucleic acid molecule to (i) an oligonucleotide barcode of the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules; and extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each comprising the first molecular label and the second molecular label.

[0024] The disclosure herein includes a method for determining the number of nucleic acid targets in a sample. In some embodiments, the method includes contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; and (i) combining the complement of the target binding region of each barcoded nucleic acid molecule with a plurality of oligonucleotide barcodes. the method includes hybridizing (i) an oligonucleotide barcode among the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules; extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a first molecular label and a second molecular label; and determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with the plurality of extended barcoded nucleic acid molecules, or products thereof.

[0025] The disclosure herein includes a method for determining the number of nucleic acid targets in a sample. In some embodiments, the method includes the steps of: contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; and interfering the complement of the target binding region of each barcoded nucleic acid molecule with (i) the plurality of oligonucleotide barcodes. the method further comprises hybridizing (i) an oligonucleotide barcode among the plurality of barcoded nucleic acid molecules, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules; extending the 3' ends of the plurality of barcoded nucleic acid molecules to produce a plurality of extended barcoded nucleic acid molecules, each of which comprises the first molecular label and the second molecular label; amplifying the plurality of extended barcoded nucleic acid molecules to produce a plurality of single-labeled nucleic acid molecules, each of which comprises the first molecular label or the second molecular label; and determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels having distinct sequences associated with the plurality of single-labeled nucleic acid molecules.

[0026] The present disclosure includes kits. In some embodiments, the kits include a plurality of oligonucleotide barcodes, each of the plurality of oligonucleotide barcodes comprising a molecular label and a target binding region, and at least 10 of the plurality of oligonucleotide barcodes comprising different molecular label sequences; a reverse transcriptase; a template switching oligonucleotide comprising the target binding region, or a portion thereof; and a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

[0027] definition Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.See, for example, 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 the purposes of this disclosure, the following terms are defined below.

[0028] As used herein, the term "adapter" may refer to a sequence for facilitating amplification or sequencing of an associated nucleic acid. The associated nucleic acid may include a target nucleic acid. The associated nucleic acid may include one or more of a spatial label, a target label, a sample label, an indexing 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 known sequences at the 5' and 3' ends, the known sequences may be the same or different sequences. The adapters positioned at the 5' and / or 3' ends of a polynucleotide may be capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, the adapter may include a universal sequence. The universal sequence may be a region of nucleotide sequence that is common to two or more nucleic acid molecules. The two or more nucleic acid molecules may also have regions of different sequences. Thus, for example, the 5' adapters can contain identical and / or universal nucleic acid sequences, and the 3' adapters can contain identical and / or universal sequences. The presence of a universal sequence in different members of a plurality of nucleic acid molecules can enable 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 can be present in different members of a collection of nucleic acid molecules, enabling 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 includes a sequence that can hybridize to such a universal sequence. A molecule having a target nucleic acid sequence can be modified to add universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of different target nucleic acid sequences.The one or more universal primers bound to the target nucleic acid may provide a site for hybridization of the universal primer. The one or more universal primers bound to the target nucleic acid may be the same or different from each other.

[0029] As used herein, the term "associated" or "associated with" can mean that two or more species are identifiable as being located together at a time. Association can mean that two or more species are or were located in similar containers. Association can also be an informational association. For example, digital information about two or more species can be stored and used to determine that one or more of the species were located together at a time. 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 also be a covalent bond between a target and a label. Association can include hybridization between two molecules (e.g., a target molecule and a label).

[0030] 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 to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," in which only some of the nucleotides bind, or complete, in which there is total complementarity between the single-stranded molecules. A first nucleotide sequence can be referred to as the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be referred to as the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, a "complementary" sequence can refer to the "complement" or "reverse complement" of a sequence. It is understood from this disclosure that when a molecule is capable of hybridizing to another molecule, it may be complementary or partially complementary to the hybridizing molecule.

[0031] 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 methodology, which can be inherently probabilistic, converts the problem of molecular counting into a series of yes / no digital questions about the detection of a predefined set of labels, one of the location and identification of identical molecules. As used herein, the term "label" or "labels" may refer to a nucleic acid code associated with a target in a sample. The label may be, for example, a nucleic acid label. The label may be a wholly or partially amplifiable label. The label may be a wholly or partially sequenceable label. The label may be a portion of a naturally occurring nucleic acid that can be identified as distinct. The label may be a known sequence. The label may comprise a junction of a nucleic acid sequence, e.g., a junction of a naturally occurring and non-natural sequence. As used herein, the term "label" may be used interchangeably with the terms "index," "tag," or "label tag." The label may carry information. For example, in various embodiments, the label can be used to determine the identity of the sample, the source of the sample, the identity of the cell, and / or the target.

[0032] As used herein, the term "non-depletion reservoir" can refer to a pool of barcodes (e.g., stochastic barcodes) composed of a large number of different labels. The non-depletion reservoir can contain a large number of different barcodes, so that when the non-depletion reservoir is associated with a pool of targets, each target is likely to associate with a unique barcode. The uniqueness of each labeled target molecule can be determined by random selection statistics and depends on the copy number of identical target molecules in the population compared to the diversity of the labels. The size of the resulting labeled target molecule can be determined by the stochastic nature of the barcoding process, and analysis of the number of detected barcodes then allows for calculation of the number of target molecules present in the original population or sample. If the ratio of the number of target molecules present to the number of unique barcodes is low, the labeled target molecule is highly unique (i.e., the probability that more than one target molecule will be labeled with a given label is very low).

[0033] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid may comprise nucleotides. A nucleic acid may be exogenous or endogenous to a cell. A nucleic acid may exist in a cell-free environment. A nucleic acid may be a gene or a fragment thereof. A nucleic acid may be DNA. A nucleic acid may be RNA. A nucleic acid may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked 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. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0034] Nucleic acids may contain one or more modifications (e.g., base modifications, backbone modifications) to result in nucleic acids with new or enhanced properties (e.g., improved stability). Nucleic acids may also contain nucleic acid affinity tags. Nucleosides may be base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides may be nucleosides further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides containing a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. When forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound may then be further joined to form a circular compound, although linear compounds are generally preferred. In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold in such a manner as to produce fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups may generally be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone may be a 3' and 5' phosphodiester linkage.

[0035] The nucleic acids can contain modified backbones and / or modified internucleoside linkages. Modified backbones can 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 therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, e.g., 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, e.g., 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs, and those with reverse polarity, wherein one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0036] 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 heteroatom or heterocyclic internucleoside linkages, including those with 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 CH moieties.

[0037] Nucleic acids may include nucleic acid mimetics. The term "mimetics" 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 may also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In PNA, the sugar backbone of a polynucleotide may be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides may 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 may contain two or more linked aminoethylglycine units, thereby providing the PNA with an amide-containing backbone. The heterocyclic base moiety may be directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Nucleic acid can include morpholino backbone structure.For example, nucleic acid can include 6-membered morpholino ring instead of ribose ring.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace phosphodiester linkage.

[0038] Nucleic acids can contain linked morpholino units (e.g., morpholino nucleic acids) with heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds may have fewer undesirable interactions with intracellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be attached using different linking groups. A further class of polynucleotide mimics can be called 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 to synthesize oligomeric compounds 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 stability similar to that of native complexes. Further modifications include locked nucleic acids (LNAs), in which a 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. 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.

[0039] Nucleic acids can also include nucleobase (often simply referred to as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases can 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 alkyl derivatives of the pyrimidine base, 6-azouracil, cytosine, 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, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidines (2H-pyrimido(4,5-b)indol-2-one), and pyridoindole cytidines (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one). As used herein, the term "sample" can refer to a composition that contains a target. Samples suitable for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.

[0040] As used herein, the term "sample collection device" or "device" may refer to a device capable of collecting a section of a sample and / or placing the section on a substrate. A sample device may refer to, for example, a fluorescence activated cell sorter (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a microtome. As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which multiple barcodes (e.g., stochastic barcodes) can be attached. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) to which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support may comprise discrete particles that may be spherical (e.g., microspheres) or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. Beads may be non-spherical in shape. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead."

[0041] As used herein, the term "stochastic barcode" may refer to a polynucleotide sequence comprising a label of the present disclosure. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0042] As used herein, the term "gene-specific stochastic barcode" may refer to a polynucleotide sequence that includes a label and a gene-specific target binding region. The stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. The stochastic barcode may be used to quantify a target in a sample. The stochastic barcode may be used to control errors that may occur after the label is associated with the target. For example, the stochastic barcode may be used to evaluate amplification or sequencing errors. The stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target. As used herein, the term "stochastic barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a Poisson recursion strategy to associate labels and quantify the labels associated with targets. As used herein, the term "stochastic barcoding" can be used interchangeably with "stochastic labeling."

[0043] As used herein, the term "target" may refer to a composition that can be associated with a barcode (e.g., a stochastic barcode). Exemplary targets suitable for analysis by the methods, devices, and systems of the present disclosure include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. Targets may be single-stranded or double-stranded. In some embodiments, targets may be proteins, peptides, or polypeptides. In some embodiments, targets are lipids. As used herein, "target" may be used interchangeably with "species."

[0044] As used herein, the term "reverse transcriptase" can refer to a group of enzymes that have reverse transcriptase activity (i.e., catalyze the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include the Lactococcus lactis LI.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases include the numerous classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity-generating retroelements, among others).

[0045] The terms "universal adapter primer," "universal primer adapter," or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can hybridize to a barcode (e.g., a stochastic barcode) and be used to generate a gene-specific barcode. The universal adapter sequence can be, for example, a known sequence that is universal across all barcodes used in the methods of the present disclosure. For example, when multiple targets are labeled using the methods disclosed herein, each of the target-specific sequences can be linked to the same universal adapter sequence. In some embodiments, two or more universal adapter sequences can be used in the methods disclosed herein. For example, when multiple targets are labeled using the methods disclosed herein, at least two of the target-specific sequences are linked to different universal adapter sequences. The universal adapter primer and its complement can be included in two oligonucleotides, one of which contains the target-specific sequence and the other of which contains the barcode. For example, the universal adapter sequence can be part of an oligonucleotide that contains a target-specific sequence to generate a nucleotide sequence that is complementary to the target nucleic acid. A second oligonucleotide comprising the barcode and the complementary sequence of the universal adapter sequence can hybridize with the nucleotide sequence to generate a target-specific barcode (target-specific stochastic barcode). In some embodiments, the universal adapter primer has a different sequence than the universal PCR primer used in the disclosed methods.

[0046] Barcode Barcoding, e.g., stochastic barcoding, is described, for example, in U.S. Patent Application Publication No. US2015 / 0299784, International Publication No. WO2015 / 031691, and Fu et al., Proc Natl Acad Sci USA 2011 May 31;108(22):9026-31, the contents of which are incorporated herein in their entireties. In some embodiments, the barcodes disclosed herein may be stochastic barcodes, which may be polynucleotide sequences that can be used to stochastically label (e.g., barcode, tag) targets. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences of the stochastic barcode to the number of occurrences of any of the targets to be labeled can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, or can be approximately these values or such numbers or ranges. The targets may be mRNA species that include mRNA molecules with identical or nearly identical sequences. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences of the stochastic barcode to the number of occurrences of any of the targets to be labeled is at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1. The barcode sequences of a stochastic barcode may be referred to as molecular labels.

[0047] A barcode, e.g., a stochastic barcode, can include one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. FIG. 1 shows an exemplary barcode 104 having a spatial label. The barcode 104 can include a 5' amine that can link the barcode to a solid support 105. The barcode can include a universal label, a dimensional label, a spatial label, a cell label, and / or a molecular label. The order of different labels (including, but not limited to, the universal label, the dimensional label, the spatial label, the cell label, and the molecular label) within the barcode can vary. For example, as shown in FIG. 1, the universal label can be the 5'-most label and the molecular label can be the 3'-most label. The spatial label, the dimensional label, and the cell label can be in any order. In some embodiments, the universal label, the spatial label, the dimensional label, the cell label, and the molecular label are in any order. The barcode can include a target binding region. The target binding region can interact with a target (e.g., target nucleic acid, RNA, mRNA, DNA) in a sample. For example, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. In some cases, the labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence) can be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides apart.

[0048] Labels, e.g., cellular labels, can include a set of unique nucleic acid subsequences of defined length, e.g., seven nucleotides each (equivalent to the number of bits used in some Hamming error-correcting codes), that can be designed to provide error-correcting capabilities. An error-correcting subsequence set including seven-nucleotide sequences can be designed so that any pairwise combination of sequences within the set exhibits a defined "genetic distance" (or number of mismatched bases); for example, an error-correcting subsequence set can be designed to exhibit a genetic distance of three nucleotides. In this case, consideration of the error-correcting sequences in the sequence dataset of the labeled target nucleic acid molecule (described in more detail below) can enable amplification or sequencing errors to be detected or corrected. In some embodiments, the length of the nucleic acid subsequences used to create the error-correcting code can vary, e.g., be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or a number or range between any two of these values, or a length of approximately these values or such a number or range of nucleotides. In some embodiments, nucleic acid subsequences of other lengths may be used to create error-correcting codes.

[0049] The barcode may include a target binding region. The target binding region may interact with a target in the sample. The target may be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNAs each containing a poly(A) tail, or any combination thereof. In some embodiments, the multiple targets may include deoxyribonucleic acid (DNA).

[0050] In some embodiments, the target binding region may include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more of the labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence (e.g., molecular label)) may be spaced from another one or two of the remaining labels of the barcode by a spacer. The spacer may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, none of the labels of the barcode are spaced apart by a spacer.

[0051] Universal Signage A barcode may include one or more universal labels. In some embodiments, the one or more universal labels may be the same for all barcodes in a set of barcodes bound to a given solid support. In some embodiments, the one or more universal labels may be the same for all barcodes bound to multiple beads. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer. The sequencing primer can be used to sequence barcodes that include a universal label. The sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a PCR primer. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer and a PCR primer. The nucleic acid sequence of a universal label that can hybridize to a sequencing primer or a PCR primer may be referred to as a primer binding site. The universal label may include a sequence that can be used to initiate transcription of the barcode. The universal label may include a sequence that can be used to extend the barcode or a region within the barcode. The universal label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values or such number or range of nucleotides in length. For example, the universal label can include at least about 10 nucleotides. The universal label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal label sequence to allow the barcode to be cleaved from the support.

[0052] dimensional indicator A barcode may include one or more dimensional labels. In some embodiments, a dimensional label may include a nucleic acid sequence that provides information about the dimension in which labeling (e.g., stochastic labeling) occurred. For example, a dimensional label can provide information about the time a target was barcoded. A dimensional label may be associated with the time of barcoding (e.g., stochastic barcoding) in a sample. A dimensional label may be activated at the time of labeling. Different dimensional labels may be activated at different time points. A dimensional label provides information about the order in which a target, a group of targets, and / or a sample was barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. Cells may be pulsed again with a barcode (e.g., a stochastic barcode) in the G1 phase of the cell cycle. Cells may be pulsed again with a barcode in the S phase of the cell cycle, and so on. The barcode in each pulse (e.g., each stage of the cell cycle) may include a different dimensional label. In this way, dimensional labels provide information about which targets were labeled at which stage of the cell cycle. Dimensional labels can interrogate many different biological times. Exemplary biological time periods include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation.In another example, a sample (e.g., cell, cell population) can be stochastically labeled before and / or after treatment with a drug and / or therapy.The change in copy number of distinct targets can indicate the response of the sample to the drug and / or therapy.

[0053] Dimensional labels may be activatable. Activatable dimensional labels can be activated at a specific time. Activatable labels can, for example, be continuously activated (e.g., not turned off). Activatable dimensional labels can, for example, be reversibly activatable (e.g., they can be turned on and turned off). Dimensional labels may be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Dimensional labels may be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, dimensional labels can be activated by fluorescence, light, chemical events (e.g., cleavage, ligation of another molecule, addition of a modification (e.g., pegylation, sumoylation, acetylation, deacetylation, demethylation), photochemical events (e.g., photocaging), and introduction of unnatural nucleotides.

[0054] In some embodiments, the dimension labels may be the same for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., a bead), but may be different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 60% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 95% of the barcodes on the same solid support may comprise the same dimension label.

[0055] On multiple solid supports (e.g., beads), 6Many unique dimension label sequences, even more than 10, may be presented. Dimension labels can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values or such number or range of nucleotides. Dimension labels can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Dimension labels can comprise from about 5 to about 200 nucleotides. Dimension labels can comprise from about 10 to about 150 nucleotides. Dimension labels can comprise from about 20 to about 125 nucleotides in length.

[0056] spatial sign The barcode may include one or more spatial labels. In some embodiments, the spatial label may include a nucleic acid sequence that provides information about the spatial orientation of the target molecule associated with the barcode. The spatial label may be associated with a coordinate in the sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed relative to a substrate. The spatial label may refer to a two-dimensional or three-dimensional grid. The coordinate may be fixed relative to a landmark. The landmark may be identifiable in space. The landmark may be a structure that can be imaged. The landmark may be a biological structure, e.g., an anatomical landmark. The landmark may be a cellular landmark, e.g., an organelle. The landmark may be a non-natural landmark, e.g., an identifiable identifier, e.g., a color code, a barcode, a magnetic property, a fluorescent property, a radioactive property, or a structure with a unique size or shape. The spatial label may be associated with a physical compartment (e.g., a well, a container, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more locations in space.

[0057] Spatial labels may be the same for all barcodes attached to a given solid support (e.g., beads), but may be different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values or such a number or range. In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support may contain the same spatial label. In some embodiments, at least 95% of the barcodes on the same solid support may contain the same spatial label.

[0058] On multiple solid supports (e.g., beads), 6 Many unique spatial marker sequences, even more than 10, may be presented. Spatial markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values or such number or range of nucleotides. Spatial markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Spatial markers can comprise from about 5 to about 200 nucleotides. Spatial markers can comprise from about 10 to about 150 nucleotides. Spatial markers can comprise from about 20 to about 125 nucleotides in length.

[0059] cell labeling A barcode (e.g., a stochastic barcode) may include one or more cell labels. In some embodiments, the cell label may include a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell. In some embodiments, the cell label is the same for all barcodes attached to a given solid support (e.g., a bead) but different for different solid supports (e.g., a bead). In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of the barcodes on the same solid support may contain the same cell label. As another example, at least 95% of the barcodes on the same solid support may contain the same cell label.

[0060] On multiple solid supports (e.g., beads), 6 Many unique cell marker sequences, even more than 10, may be presented. Cell markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values or such number or range of nucleotides. Cell markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. For example, a cell marker can comprise from about 5 to about 200 nucleotides. As another example, a cell marker can comprise from about 10 to about 150 nucleotides. As yet another example, a cell marker can comprise from about 20 to about 125 nucleotides in length.

[0061] Barcode sequence The barcode may include one or more barcode sequences. In some embodiments, the barcode sequence may include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. The barcode sequence includes a nucleic acid sequence that provides a counter (e.g., provides a rough approximation) for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target binding region). In some embodiments, a diverse set of barcode sequences is attached to a given solid support (e.g., a bead). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 There may be at least 10 unique molecular label sequences, or a number or range between or about any two of these values. For example, the plurality of barcodes may include about 6561 barcode sequences with distinct sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences with distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces or 10 9 There may be multiple unique barcode sequences. The unique molecular label sequence may be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular label sequence is partially or entirely encompassed by a particle (e.g., a hydrogel bead).

[0062] The length of the barcode may vary in different implementations. For example, the barcode may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, or may be approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, in nucleotide length. As another example, the barcode may be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0063] molecular label A barcode (e.g., a stochastic barcode) may include one or more molecular labels. A molecular label may include a barcode sequence. In some embodiments, a molecular label may include a nucleic acid sequence that provides identification of information regarding the specific type of target nucleic acid species hybridized to the barcode. A molecular label includes a nucleic acid sequence that provides a counter for the specific occurrence of a target nucleic acid species hybridized to the barcode (e.g., a target binding region). In some embodiments, a diverse set of molecular labels is attached to a given solid support (e.g., beads). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 or a number or range between any two of these values, or about 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9There may be at least 10 unique molecular label sequences, or a number or range between any two of these values. For example, the plurality of barcodes may include about 6561 molecular labels with distinct sequences. As another example, the plurality of barcodes may include about 65536 molecular labels with distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There can be a number of unique molecular beacon sequences. A barcode with a unique molecular beacon sequence can be attached to a given solid support (e.g., a bead).

[0064] For barcoding using multiple stochastic barcodes (e.g., stochastic barcoding), the ratio of the number of distinct molecular beacon sequences to the number of occurrences of any of the targets can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or or a number or range between any two of these values, or may be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target may be an mRNA species containing mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular label sequences to the number of occurrences of any of the targets is at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0065] A molecular label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or a number or range of nucleotides approximately equal to or equal to these values or such number or range. A molecular label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0066] Target binding region The barcode may include one or more target binding regions, e.g., capture probes. In some embodiments, the target binding region may hybridize with a target of interest. In some embodiments, the target binding region may include a nucleic acid sequence that specifically hybridizes to a target (e.g., a target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), e.g., a specific gene sequence. In some embodiments, the target binding region may include a nucleic acid sequence that can bind (e.g., hybridize) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region may include a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be ligated to any nucleic acid molecule that includes a sequence complementary to the restriction site overhang.

[0067] In some embodiments, the target binding region may include a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence may refer to a sequence that can bind to multiple target nucleic acids independently of the specific sequence of the target nucleic acid. For example, the target binding region may include a random multimer sequence or an oligo(dT) sequence that hybridizes to the poly(A) tail on an mRNA molecule. The random multimer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or any longer multimer sequence of any length. In some embodiments, the target binding region is the same for all barcodes bound to a given bead. In some embodiments, the target binding regions of multiple barcodes bound to a given bead may include two or more different target binding sequences. The target binding region can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between or about any two of these values. The target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.

[0068] In some embodiments, the target binding region can comprise an oligo(dT) that can hybridize with mRNA containing polyadenylated ends. The target binding region can be gene-specific. For example, the target binding region can be configured to hybridize with a specific region of the target. The target binding region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a number or range between any two of these values, or approximately these values or such number or range of nucleotides in length. The target binding region can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The target binding region can be about 5 to 30 nucleotides in length. When a barcode includes a gene-specific target binding region, the barcode may be referred to herein as a gene-specific barcode.

[0069] Orientation Characteristics A stochastic barcode (e.g., a stochastic barcode) may include one or more orientation properties that can be used to orient (e.g., align) the barcode. The barcode may include a moiety for isoelectric focusing. Different barcodes may include different isoelectric focusing points. When these barcodes are introduced into a sample, the sample may undergo isoelectric focusing to orient the barcodes in a known manner. In this manner, the orientation properties can be used to develop a known map of the barcodes in the sample. Exemplary orientation properties can include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, a barcode with an orientation property for self-assembly may self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0070] affinity properties A barcode (e.g., a stochastic barcode) can include one or more affinity features. For example, a spatial label can include an affinity feature. Affinity features can include chemical and / or biological moieties that can facilitate binding of the barcode to another entity (e.g., a cellular receptor). For example, an affinity feature can include an antibody, e.g., an antibody specific to a particular moiety (e.g., a receptor) on a sample. In some embodiments, the antibody can direct the barcode to a particular cell type or molecule. Targets at and / or near a particular cell type or molecule can be labeled (e.g., stochastically labeled). In some embodiments, the affinity feature can provide spatial information in addition to the nucleotide sequence of the spatial label, as the antibody can direct the barcode to a specific location. The antibody can be a therapeutic antibody, e.g., a monoclonal or polyclonal antibody. The antibody can be humanized or chimeric. The antibody can be a naked antibody or a fusion antibody. An antibody can be a full-length (i.e., naturally occurring or formed 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.

[0071] An antibody fragment can be, for example, a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, 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 (CD8, CD34, CD45), and therapeutic antibodies.

[0072] Universal Adapter Primer A barcode can include one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, can include a universal adapter primer. The universal adapter primer can refer to a universal nucleotide sequence across all barcodes. The universal adapter primer can be used to construct a gene-specific barcode. The universal adapter primer can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides in length, or a number or range between any two of these, or approximately these values or such number or range of nucleotides. The universal adapter primer can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The universal adapter primer can be 5 to 30 nucleotides in length.

[0073] Linker When a barcode includes more than one type of label (e.g., more than one cell label or more than one barcode sequence, e.g., one molecular label), the labels may be interspersed with linker label sequences. The linker label sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. The linker label sequence may be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. In some cases, the linker label sequence is 12 nucleotides in length. The linker label sequence can be used to facilitate the synthesis of the barcode. The linker label may include an error-correcting (e.g., Hamming) code.

[0074] solid support In some embodiments, the barcodes disclosed herein, e.g., stochastic barcodes, may be associated with a solid support. The solid support may be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences, e.g., molecular labels of stochastic barcodes (e.g., first barcode sequences) of a plurality of barcodes (e.g., a first plurality of barcodes) on a solid support, differ by at least one nucleotide. The cellular labels of barcodes on the same solid support may be the same. The cellular labels of barcodes on different solid supports may differ by at least one nucleotide. For example, a first cellular label of a first plurality of barcodes on a first solid support may have the same sequence, and a second cellular label of a second plurality of barcodes on a second solid support may have the same sequence. The first cellular label of a first plurality of barcodes on a first solid support and the second cellular label of a second plurality of barcodes on a second solid support may differ by at least one nucleotide. The cellular labels may be, for example, about 5 to 20 nucleotides in length. The barcode sequence can be, for example, about 5-20 nucleotides in length. The synthetic particle can be, for example, a bead.

[0075] The beads can be, for example, silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic materials, ceramic, plastic, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof.

[0076] In some embodiments, the beads can be polymer beads, such as deformable beads or gel beads (e.g., gel beads from 10X Genomics (San Francisco, CA)) functionalized with barcodes or stochastic barcodes. In some implementations, the gel beads can comprise a polymer-based gel. Gel beads can be generated, for example, by encapsulating one or more polymer precursors into droplets. Gel beads can be generated when the polymer precursors are exposed to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0077] In some embodiments, the particles may be disintegrable (e.g., dissolvable, degradable). For example, polymer beads may dissolve, melt, or decompose under desired conditions, for example. The desired conditions may include environmental conditions. The desired conditions may result in the dissolution, melting, or decomposition of the polymer beads in a controlled manner. Gel beads may dissolve, melt, or decompose due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electrical stimulus, a light stimulus, or any combination thereof.

[0078] Analytes and / or reagents, e.g., oligonucleotide barcodes, may be linked / immobilized, for example, to the interior surface of a gel bead (e.g., the interior accessible through diffusion of the oligonucleotide barcodes and / or the material used to generate the oligonucleotide barcodes) and / or to the exterior surface of a gel bead or any other microcapsule described herein. Linkage / immobilization may be via any form of chemical bond (e.g., covalent bond, ionic bond) or physical phenomenon (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the linkage / immobilization of reagents to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical crosslinker, including those described herein). Upon application of a stimulus, the labile moiety can be cleaved, releasing the immobilized reagent. In some embodiments, the labile moiety is a disulfide bond. For example, in cases where an oligonucleotide barcode is immobilized to a gel bead via a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the bead. The labile moiety may be included as part of the gel bead or microcapsule, as part of a chemical linker connecting the reagent or analyte to the gel bead or microcapsule, and / or as part of the reagent or analyte. In some embodiments, at least one barcode of the plurality of barcodes may be immobilized to a particle, partially immobilized to a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof.

[0079] In some embodiments, the gel beads may comprise a wide variety of different polymers, including, but not limited to, polymers, thermosensitive polymers, light-sensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. Polymers can include, but are not limited to, materials such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethylammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthalaldehyde) (PTHF), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic-co-glycolic acid) (PLGA).

[0080] A number of chemical stimuli can be used to trigger bead collapse, dissolution, or degradation. Examples of these chemical changes include, but are not limited to, pH-mediated changes to the bead wall, bead wall collapse via chemical cleavage of cross-links, triggering bead wall depolymerization, and bead wall switching reactions. Bulk changes can also be used to trigger bead collapse.

[0081] Bulk or physical changes to microcapsules through various stimuli also offer many advantages in designing capsules for releasing reagents. Bulk or physical changes occur on a macroscopic scale, with bead rupture being the result of mechanical-physical forces induced by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in bead wall porosity.

[0082] Biological stimuli can also be used to trigger bead disintegration, dissolution, or degradation. Generally, biological triggers resemble chemical triggers, but many examples use biomolecules, or molecules commonly found in biological systems, such as enzymes, peptides, sugars, fatty acids, and nucleic acids. For example, beads can contain polymers with peptide crosslinks that are susceptible to cleavage by specific proteases. More specifically, one example can include microcapsules containing GFLGK peptide crosslinks. Addition of a biological trigger, such as the protease cathepsin B, cleaves the peptide crosslinks in the shell wall, releasing the contents of the bead. In other cases, the protease can be heat-activated. In another example, beads contain a shell wall containing cellulose. Addition of the hydrolytic enzyme chitosan serves as a biological trigger to cleave the cellulose bonds, depolymerize the shell wall, and release its contents.

[0083] Beads can also be induced to release their contents upon application of a thermal stimulus. A change in temperature can cause various changes in the beads. A change in heat can cause the beads to melt, causing the bead walls to collapse. In other cases, heat can increase the internal pressure of the beads' internal components, causing the beads to collapse or explode. In yet other cases, heat can transform the beads into a compressed, dehydrated state. Heat can also act on heat-sensitive polymers within the bead walls, causing the beads to collapse. By including magnetic nanoparticles in the bead walls of the microcapsules, it is possible to trigger the collapse of the beads and guide the beads in an array. The devices of the present disclosure can include magnetic beads for either purpose. In one example, by incorporating Fe3O4 nanoparticles into polyelectrolyte-containing beads, collapse is triggered in the presence of an oscillating magnetic field stimulus. Beads can also be disintegrated, dissolved, or decomposed as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrosensitive beads can trigger both bead disintegration and other functions, such as alignment in an electric field, electrical conduction, or redox reactions. In one example, beads containing electrosensitive materials are aligned in an electric field to control the release of internal reagents. In another example, an electric field can induce redox reactions within the bead wall itself, which can increase porosity.

[0084] Light stimulation can also be used to disrupt the beads. Numerous optical triggers are possible, including systems using various molecules, such as nanoparticles and chromophores, that can absorb photons of specific wavelengths. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of SiO2-coated polyelectrolyte capsules can result in the collapse of the bead wall. In yet another example, photoswitchable materials, such as azobenzene groups, can be incorporated into the bead wall. Upon application of UV or visible light, chemicals such as these absorb photons and undergo reversible cis-to-trans isomerization. In this embodiment, the incorporation of a photoswitch results in a bead wall that can collapse or become more porous upon application of a light trigger. For example, in a non-limiting example of barcoding (e.g., stochastic barcoding) shown in FIG. 2, after cells, e.g., single cells, are introduced into multiple microwells of a microwell array in block 208, beads can be introduced into multiple microwells of the microwell array in block 212. Each microwell may contain one bead. The beads may contain multiple barcodes. The barcodes may include 5' amine regions attached to the beads. The barcodes may include a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0085] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a bead). The barcodes associated with the solid support may comprise a barcode sequence selected from a group comprising at least 100 or 1000 barcode sequences, each having a unique sequence. In some embodiments, different barcodes associated with the solid support may comprise barcodes with different sequences. In some embodiments, a percentage of the barcodes associated with the solid support comprise the same cell marker. For example, the percentage may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values or such a number or range. As another example, the percentage may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support may have the same cell label. Barcodes associated with different solid supports may have different cell labels selected from a group comprising at least 100 or 1000 cell labels having unique sequences.

[0086] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a bead). In some embodiments, barcoding a plurality of labels in a sample can be performed using a solid support comprising a plurality of synthetic particles associated with a plurality of barcodes. In some embodiments, the solid support may comprise a plurality of synthetic particles associated with a plurality of barcodes. The spatial labeling of the plurality of barcodes on different solid supports may differ by at least one nucleotide. The solid support may comprise a plurality of barcodes, for example, in two or three dimensions. The synthetic particles may be beads. The beads may be silica gel beads, controlled-pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support may include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support may be free-floating. In some embodiments, the solid support may be embedded in a semi-solid or solid array. The barcodes may not be associated with a solid support. The barcodes may be individual nucleotides. The barcode may be associated with the substrate.

[0087] As used herein, the terms "tethered," "attached," and "immobilized" are used interchangeably and can refer to covalent or non-covalent means for attaching a barcode to a solid support. Any of a variety of different solid supports can be used to attach pre-synthesized barcodes or as a solid support for in situ solid phase synthesis of barcodes. In some embodiments, the solid support is a bead. The bead may comprise one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures that can immobilize nucleic acids (e.g., covalently or non-covalently). The bead may be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. The bead may be or comprise a discrete particle that is spherical (e.g., a microsphere), or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. In some embodiments, the bead may be non-spherical in shape.

[0088] The beads may comprise a variety of materials, including, but not limited to, paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe3O4, magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof. In some embodiments, the beads (e.g., the beads to which the label is attached) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0089] Some embodiments disclosed herein include one or more particles (e.g., beads). Each of the particles may include a plurality of oligonucleotides (e.g., barcodes). Each of the plurality of oligonucleotides may include a barcode sequence (e.g., a molecular label sequence), a cell label, and a target binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequence of each of the plurality of oligonucleotides may be the same. The cell label sequences of oligonucleotides on different particles may be different so that the oligonucleotides on different particles can be identified. The number of different cell label sequences may vary in different implementations. In some embodiments, the number of cell labeling sequences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 In some embodiments, the number of cell labeling sequences is at least or at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces or 10 9In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the plurality of particles comprise oligonucleotides having the same cellular sequence. In some embodiments, up to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of the particles comprise oligonucleotides with the same cellular sequence. In some embodiments, none of the particles in the plurality have the same cellular labeling sequence.

[0090] The multiple oligonucleotides on each particle can include different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9In some embodiments, the number of barcode sequences may be at least or at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces or 10 9 For example, at least 100 of the plurality of oligonucleotides may contain different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or range between any two of these values, or more of the plurality of oligonucleotides may contain different barcode sequences. Some embodiments provide a plurality of particles comprising barcodes. In some embodiments, the ratio of occurrence (or copy or number) of the target to be labeled to the different barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or a microparticle.

[0091] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or range between any two of these values, or can be approximately these values or such numbers or ranges.

[0092] The diameter of the beads may be related to the diameter of the wells of the substrate. In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between or about any two of these values, longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or range between any two of these values, or a number or range between or about these values or any such number or range, longer or shorter than the diameter of the cells. In some embodiments, the diameter of the beads may be at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the cells.

[0093] The beads may be bound and / or embedded in a substrate. The beads may be bound and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial location of the beads within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial labels present in barcodes on the beads, which can serve as location addresses. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), 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 BcMag™ carboxyl-terminated magnetic beads.

[0094] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one fluorescent optical channel or multiple optical channels. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be identifiable. For example, the beads can be imaged using a camera. The beads can have a detectable code associated with them. For example, the beads can include a barcode. The beads can change size, for example, due to swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible.

[0095] The solid support (e.g., beads) can be visualized. The solid support can include a visualization tag (e.g., a fluorescent dye). The solid support (e.g., beads) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging of the beads. A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as "functionalized" if it contains a linker, scaffold, building block, or other reactive moiety attached thereto, but can be "non-functionalized" if it lacks such a reactive moiety attached thereto. A solid support can be freely used in solution, for example, in a microtiter well format, in a flow-through format, for example, in a column, or in a dipstick.

[0096] The solid support may comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. The solid support may take the form of a resin, gel, microsphere, or other geometric configuration. The solid support may comprise a silica chip, microparticle, nanoparticle, plate, array, caliper, flat support, such as a glass fiber filter, glass surface, metal surface, metal surface (steel, gold / silver, aluminum, silicon, and copper), glass support, plastic support, silicon support, chip, filter, membrane, microwell plate, slide, multiwell plate, or plastic material (e.g., made of polyethylene, polypropylene, polyamide, polyvinylidene difluoride), including membrane, and / or wafer, comb, pin, or needle (e.g., an array of pins suitable for combinatorial synthesis or analysis), or an array of holes or nanoliter wells on a flat surface, such as a wafer (e.g., a silicon wafer), a wafer with holes or without a filter bottom, or beads. The solid support can include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix can be capable of penetrating intracellular spaces (e.g., around organelles). The polymer matrix can be capable of being pumped through the circulatory system.

[0097] Substrates and microwell arrays As used herein, a substrate may refer to a type of solid support. A substrate may refer to a solid support that may include a barcode or stochastic barcode of the present disclosure. A substrate may include, for example, multiple microwells. For example, a substrate may be a well array including two or more microwells. In some embodiments, a microwell may include a small reaction chamber with a defined volume. In some embodiments, a microwell may incorporate one or more cells. In some embodiments, a microwell may incorporate only one cell. In some embodiments, a microwell may incorporate one or more solid supports. In some embodiments, a microwell may incorporate only one solid support. In some embodiments, a microwell incorporates a single cell and a single solid support (e.g., a bead). A microwell may include a barcode reagent of the present disclosure.

[0098] Barcoding methods The present disclosure provides a method for estimating the number of distinct targets in distinct locations of a body sample (e.g., tissue, organ, tumor, cell). The method may include placing a barcode (e.g., a stochastic barcode) in proximity to the sample, lysing the sample, associating distinct targets with the barcode, amplifying the targets, and / or digitally counting the targets. The method may further include analyzing and / or visualizing information obtained from the spatial labeling of the barcode. In some embodiments, the method includes visualizing multiple targets in the sample. Mapping the multiple targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps may be created before or after barcoding (e.g., stochastically barcoding) the multiple targets in the sample. Visualizing the multiple targets in the sample may include mapping the multiple targets to a map of the sample. Mapping the multiple targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. Two-dimensional and three-dimensional maps can be created before or after barcoding multiple targets in sample.In some embodiments, two-dimensional and three-dimensional maps can be created before or after dissolving sample.Dissolving sample before or after creating two-dimensional or three-dimensional map can include heating sample, contacting sample with detergent, changing the pH of sample, or any combination thereof.

[0099] In some embodiments, barcoding the plurality of targets comprises hybridizing a plurality of barcodes to the plurality of targets to create barcoded targets (e.g., stochastically barcoded targets). Barcoding the plurality of targets may comprise generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets may be performed using a solid support comprising a plurality of barcodes (e.g., stochastic barcodes).

[0100] Contacting the sample with the barcode The present disclosure provides a method for contacting a sample (e.g., cells) with a substrate of the present disclosure. For example, a sample including a thin section of cells, an organ, or a tissue can be contacted with a barcode (e.g., a stochastic barcode). For example, the cells can be contacted by gravity flow, where they can settle and form a monolayer. The sample can be a tissue slice. The slice can be placed on a substrate. The sample can be one-dimensional (e.g., forming a planar surface). For example, the sample (e.g., cells) can be spread across the substrate by growing / culturing the cells on the substrate. When the barcode is in close proximity to the target, the target can hybridize to the barcode. The barcodes can be contacted in a non-depleting ratio so that each distinct target can associate with a distinct barcode of the present disclosure. To ensure efficient association between the target and the barcode, the target can be cross-linked to the barcode.

[0101] Cell lysis After the cells and barcodes are separated, the cells can be lysed to release the target molecule. Cell lysis can be achieved by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by thermal lysis, mechanical lysis, or optical lysis. Cells can also be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, Li-dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association between the target and the barcode, the diffusion rate of the target molecule can be changed, for example, by lowering the temperature and / or increasing the viscosity of the lysate. In some embodiments, the sample may be lysed using filter paper, which can be soaked with a lysis buffer over the filter paper, and pressure can be applied to the sample to promote lysis of the sample and hybridization of the sample's targets to the substrate.

[0102] In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris-HCl. The lysis buffer can include at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include up to about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include about 0.1 M Tris-HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may contain a salt (e.g., LiCl). The salt concentration in the lysis buffer may be at least about 0.1, 0.5, or 1 M or higher. The salt concentration in the lysis buffer may be up to about 0.1, 0.5, or 1 M or higher. In some embodiments, the salt concentration in the lysis buffer is about 0.5 M. The lysis buffer may contain a detergent (e.g., SDS, Li-dodecyl sulfate, triton X, tween, NP-40). The concentration of the detergent in the lysis buffer may be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. The concentration of surfactant in lysis buffer can be up to about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher.In some embodiments, the concentration of surfactant in lysis buffer is about 1% Li-dodecyl sulfate.The time used in the method for lysis can depend on the amount of surfactant used.In some embodiments, the more detergent used, the less time is required for lysis. The lysis buffer may contain a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1, 5, 10, 15, 20, 25, or 30 mM, or higher. The concentration of the chelating agent in the lysis buffer may be up to about 1, 5, 10, 15, 20, 25, or 30 mM, or higher. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer may contain a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM, or higher. The concentration of the reducing reagent in the lysis buffer may be up to about 1, 5, 10, 15, or 20 mM, or higher. In some embodiments, the concentration of the reducing reagent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may comprise about 0.1 M Tris-HCl (about pH 7.5), about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0103] Lysing can be performed at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysing can be performed for about 1, 5, 10, 15, 20 minutes, or longer. Lysed cells can contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules. Lysed cells can contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules.

[0104] Attaching the barcode to the target nucleic acid molecule After cell lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with the barcodes on the co-localized solid support. Association may involve hybridization of the target recognition region of the barcode to a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode may interact with the poly(A) tail of the target). Assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) used for hybridization may be selected to promote the formation of specific, stable hybrids. In some embodiments, nucleic acid molecules released from lysed cells may associate with multiple probes on a substrate (e.g., hybridize to the probes on a substrate). If the probes contain oligo(dT), mRNA molecules may hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide may act as a primer for first-strand synthesis of cDNA molecules. For example, in the non-limiting example of barcoding shown in block 216 of FIG. 2, mRNA molecules may hybridize to barcodes on beads. For example, a single-stranded nucleotide fragment can hybridize to the target binding region of the barcode.

[0105] The binding may further include ligating the target recognition region of the barcode with a portion of the target nucleic acid molecule. For example, the target binding region may include a nucleic acid sequence capable of specific hybridization to a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure may further include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to generate a restriction site overhang. The barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to connect the two fragments. For example, in a non-limiting example of barcoding shown in block 220 of Figure 2, labeled targets (e.g., target-barcode molecules) from multiple cells (or multiple samples) can then be pooled, e.g., in a tube. For example, the labeled targets can be pooled by collecting beads to which barcodes and / or target-barcode molecules are bound. Solid support-based collection of bound target-barcode molecules can be achieved by using magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can proceed within a single reaction vessel. Further processing can include, for example, reverse transcription, amplification, cleavage, dissociation, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling labeled target nucleic acid molecules from multiple cells.

[0106] Reverse transcription The present disclosure provides methods for generating target-barcode conjugates using reverse transcription (e.g., block 224 of Figure 2). The target-barcode conjugates can include a barcode and a complementary sequence of all or a portion of a target nucleic acid (i.e., a barcoded cDNA molecule, e.g., a stochastically barcoded cDNA molecule). Reverse transcription of the associated RNA molecule can occur by adding a reverse transcription primer along with a reverse transcriptase. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo(dT) primer can be 12-18 nucleotides in length, or about 12-18 nucleotides in length, and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexanucleotide primer can bind to the mRNA at various complementary sites. The target-specific oligonucleotide primer typically selectively primes the mRNA of interest.

[0107] In some embodiments, reverse transcription of the labeled RNA molecule can occur by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Typically, oligo(dT) primers are 12-18 nucleotides in length and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNAs. Random hexanucleotide primers can bind to mRNAs at various complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest. Reverse transcription can occur repeatedly to generate multiple labeled cDNA molecules. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methods can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0108] amplification One or more nucleic acid amplification reactions (e.g., block 228 of FIG. 2) can be performed to generate multiple copies of the labeled target nucleic acid molecule. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to add sequencing adapters to the nucleic acid molecule. The amplification reaction can include amplifying at least a portion of the sample label, if present. The amplification reaction can include amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction can include amplifying at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or a combination thereof. The amplification reaction may include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a range or number between any two of these values of the plurality of nucleic acids. The method may further include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of the target-barcode molecule comprising the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0109] In some embodiments, amplification can be carried out using polymerase chain reaction (PCR).As used herein, PCR can refer to the reaction for amplifying specific DNA sequences in vitro by simultaneous primer extension of complementary strands of DNA.As used herein, PCR can encompass derivatives of reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR and assembly PCR.

[0110] Amplification of labeled nucleic acids may include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiplex displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) method, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and branched extension amplification (RAM). In some embodiments, amplification does not produce circularized transcripts.

[0111] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on the labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to generate a labeled amplicon (e.g., a stochastically labeled amplicon). The labeled amplicon may be a double-stranded molecule. The double-stranded molecule may comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule may comprise a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon may be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure may include synthetic or modified nucleic acids.

[0112] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0113] Performing one or more amplification reactions may include the use of one or more primers. The one or more primers may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the multiple labeled targets (e.g., stochastically labeled targets). The one or more primers may anneal to the 3' or 5' ends of the multiple labeled targets. The one or more primers may anneal to an internal region of the multiple labeled targets. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the multiple labeled targets. The one or more primers may comprise a fixed panel of primers. The one or more primers may comprise at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more gene-specific primers.

[0114] The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. The one or more primers may include a universal primer and a custom primer. The custom primers may be designed to amplify one or more targets. The targets may comprise a subset of all nucleic acids in one or more samples. The targets may comprise a subset of all labeled targets in one or more samples. The one or more primers may include at least 96 or more custom primers. The one or more primers may include at least 960 or more custom primers. The one or more primers may include at least 9600 or more custom primers. The one or more custom primers may anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids may correspond to one or more genes.

[0115] Any amplification scheme can be used in the disclosed method. For example, in one scheme, the first PCR can amplify the molecules bound to the beads using a gene-specific primer and a primer for the sequence of universal Illumina sequencing primer 1. The second PCR can amplify the first PCR product using a nested gene-specific primer adjacent to the sequence of Illumina sequencing primer 2 and a primer for the sequence of universal Illumina sequencing primer 1. The third PCR adds P5 and P7 and a sample index to place the PCR product into an Illumina sequencing library. Sequencing using 150bp x 2 sequencing can reveal cell markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and sample indexes on index 1 read.

[0116] In some embodiments, chemical cleavage can be used to remove nucleic acids from a substrate. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from a solid support. For example, enzymes can be used to remove nucleic acids from a substrate. For example, nucleic acids can be removed from a substrate by restriction endonuclease digestion. For example, nucleic acids containing dUTP or ddUTP can be removed from a substrate using uracil-d-glycosylase (UDG) treatment. For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, for example, apurinic / apyrimidinic (AP) endonucleases, can be used to remove nucleic acids from a substrate. In some embodiments, photocleavable groups and light can be used to remove nucleic acids from a substrate. In some embodiments, a cleavable linker can be used to remove nucleic acids from a substrate. For example, the cleavable linker can comprise at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-Protein A, a photolabile linker, an acid or base labile linker group, or an aptamer. If the probe is gene-specific, the molecule can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, the nucleic acid can be amplified after being synthesized (e.g., reverse transcribed). Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acid.

[0117] In some embodiments, amplification can be performed on the substrate using, for example, bridge amplification. Homopolymer tails can be added to cDNA to generate ends compatible with bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, a primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to solid particles. When a sample containing the template nucleic acid is contacted with the particles and a single thermal cycle is performed, the template molecule anneals to the first primer, and the first primer can be extended in the forward direction by adding nucleotides to form a double-stranded molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing stage of the subsequent annealing and extension step, the complementary strand can hybridize to a second primer complementary to the segment of the complementary strand at the position removed from the first primer. Through this hybridization, the complementary strand can form a bridge between the first and second primers, immobilized by covalent bonding to the first primer and by hybridization to the second primer. During the extension step, the second primer can be extended in the opposite direction by adding nucleotides to the same reaction mixture, thereby converting the bridge into a double-stranded bridge. The next cycle then begins, and the double-stranded bridge is denatured to obtain two single-stranded nucleic acid molecules, each with one end bound to the particle surface through the first and second primers and the other end unbound. During the annealing and extension step of this second cycle, each strand can hybridize to a previously unused complementary primer on the same particle to form a new single-stranded bridge. The two previously unused primers hybridized at this point extend to convert the two new bridges into double-stranded bridges.

[0118] The amplification reaction can include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids. Amplification of the labeled nucleic acid may include PCR-based or non-PCR-based methods. Amplification of the labeled nucleic acid may include exponential amplification of the labeled nucleic acid. Amplification of the labeled nucleic acid may include linear amplification of the labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, suppression PCR, semi-suppressive PCR, and assembly PCR.

[0119] In some embodiments, the amplification of the labeled nucleic acid comprises a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ) method, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and / or branched extension amplification (RAM).

[0120] In some embodiments, the methods disclosed herein further comprise performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon may be a double-stranded molecule. The double-stranded molecule may comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule may comprise a sample tag or a molecular identifier label. Alternatively, the amplicon may be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acid of the present invention may comprise a synthetic nucleic acid or a modified nucleic acid.

[0121] In some embodiments, the method includes repeatedly amplifying a labeled nucleic acid to generate multiple amplicons. The methods disclosed herein may include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the method includes performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions. The amplification may further include adding one or more control nucleic acids to one or more samples containing the plurality of nucleic acids. The amplification may further include adding one or more control nucleic acids to the plurality of nucleic acids. The control nucleic acids may include a control label.

[0122] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile and / or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0123] Performing one or more amplification reactions may include the use of one or more primers. The one or more primers may comprise one or more oligonucleotides. The one or more oligonucleotides may comprise at least about 7 to 9 nucleotides. The one or more oligonucleotides may comprise fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the plurality of labeled nucleic acids. The one or more primers may anneal to the 3' and / or 5' ends of the plurality of labeled nucleic acids. The one or more primers may anneal to an internal region of the plurality of labeled nucleic acids. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the plurality of labeled nucleic acids. The one or more primers may comprise a fixed panel of primers. The one or more primers may comprise at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more housekeeping gene primers. The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. The one or more primers may include a universal primer and a custom primer. The custom primer may be designed to amplify one or more target nucleic acids. The target nucleic acids may comprise a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are probes attached to the array of the present disclosure.

[0124] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further includes generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of those targets. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) may be different from each other. Generating an indexed library of barcoded targets includes generating a plurality of indexed polynucleotides from the plurality of targets in the sample. For example, for an indexed library of barcoded targets including a first indexed target and a second indexed target, the labeled region of the first indexed polynucleotide may differ from the labeled region of the second indexed polynucleotide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range between, or about, or at least, or at most, any two of these values. In some embodiments, generating an indexed library of barcoded targets comprises contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides comprising a poly(T) region and a label region, and performing first-strand synthesis using a reverse transcriptase to generate single-stranded, labeled cDNA molecules, each comprising a cDNA region and a label region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an indexed library of barcoded targets may further comprise amplifying the single-stranded, labeled cDNA molecules to generate double-stranded, labeled cDNA molecules, and performing nested PCR on the double-stranded, labeled cDNA molecules to generate labeled amplicons.In some embodiments, the method may include generating adapter-labeled amplicons.

[0125] Barcoding (e.g., stochastic barcoding) can include labeling individual nucleic acid (e.g., DNA or RNA) molecules with nucleic acid barcodes or tags. In some embodiments, this includes adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can minimize PCR amplification bias. For example, adapters can be added for sequencing using next-generation sequencing (NGS). For example, sequencing results can be used to determine the sequence of cellular labels, molecular labels, and nucleotide fragments of one or more copies of the target in block 232 of FIG. 2.

[0126] 3 is a schematic diagram illustrating a non-limiting, exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), e.g., barcoded mRNAs or fragments thereof. As shown in step 1, a reverse transcription process can encode each mRNA molecule containing a unique molecular label sequence, a cellular label sequence, and a universal PCR site. In particular, the RNA molecule 302 can be reverse transcribed to generate labeled cDNA molecules 304 containing cDNA regions 306 by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to a poly(A) tail region 308 of the RNA molecule 302. Each of the barcodes 310 can include a target binding region, e.g., a poly(dT) region 312, a label region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0127] In some embodiments, the cell label sequence may comprise 3 to 20 nucleotides. In some embodiments, the molecular label sequence may comprise 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cell label, wherein the universal label is the same for the plurality of stochastic barcodes on the solid support, and the cell label is the same for the plurality of stochastic barcodes on the solid support. In some embodiments, the universal label may comprise 3 to 20 nucleotides. In some embodiments, the cell label comprises 3 to 20 nucleotides.

[0128] In some embodiments, label region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, label region 314 may include one or more of a universal label, a dimensional label, and a cell label. Barcode sequence or molecular label 318 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between any of these values, in length, or may be approximately, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in length between any of these values. A cell label 320 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between, or may be approximately, or may be at least, or may be up to, these values or such number or range of nucleotides in length. A universal label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between, or may be approximately, or may be at least, or may be up to, these values or such number or range of nucleotides in length. The universal label may be the same for multiple stochastic barcodes on a solid support, and the cell label may be the same for multiple stochastic barcodes on a solid support.A dimension label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between any of these values, in length, or may be approximately, at least, or at most, these values or numbers or ranges of nucleotides in length.

[0129] In some embodiments, label region 314 may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include approximately these values or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include at least these values or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include up to these values or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320. Each label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range between any of these values, in length, or may be approximately, at least, or at most, these values or numbers or ranges of nucleotides in length. 2 , 10 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 , or a number or range of barcodes or stochastic barcodes 310 between any of these values, or about these values or such number or range of barcodes or stochastic barcodes 310, or at least these values or such number or range of barcodes or stochastic barcodes 310, or up to these values or such number or range of barcodes or stochastic barcodes 310. Also, the set of barcodes or stochastic barcodes 310 may each contain, for example, a unique labeled region 314. The labeled cDNA molecules 304 may be purified to remove excess barcodes or stochastic barcodes 310. Purification may include Ampure bead purification.

[0130] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled in one tube and PCR amplified using a first PCR primer pool and a first universal PCR primer. Pooling is possible due to the unique label region 314. In particular, the labeled cDNA molecules 304 can be amplified to generate nested PCR-labeled amplicons 322. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, the multiplex PCR amplification can be performed in a single reaction volume using 10, 20, 40, 50, 70, 80, 90, 10, 25, 30, 35 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 1012 , 10 13 , 10 14 , 10 15 , 10 20 The amplification may utilize a number or range of multiplex primers between, or between, or at about, or at least, or at most. The amplification may include a first PCR primer pool 324 including custom primers 326A-C targeting specific genes and a universal primer 328. The custom primer 326 may hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. The universal primer 328 may hybridize to the universal PCR region 316 of the labeled cDNA molecule 304.

[0131] As shown in step 3 of Figure 3, the product from the PCR amplification in step 2 can be amplified using a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the nested PCR-labeled amplicon 322 can be further amplified by nested PCR. Nested PCR can include multiplex PCR including a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328' in a single reaction volume. Nested PCR primer pool 328 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, or may contain approximately, or at least, or at most, these values or numbers or ranges of different nested PCR primers 330. Nested PCR primers 332 may contain adaptors 334 and hybridize to regions within cDNA portion 306" of labeled amplicon 322. Universal primer 328' contains adapter 336 and can hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 generates adapter-labeled amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328' may not contain adapters 334 and 336. Instead, adapters 334 and 336 can ligate to the product of the nested PCR to generate adapter-labeled amplicon 338.

[0132] As shown in step 4, the PCR products from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 334 and 336 can be used to perform one or more additional assays on adapter-labeled amplicons 338. Adapters 334 and 336 can be hybridized with primers 340 and 342. One or more primers 340 and 342 can be PCR amplification primers. One or more primers 340 and 342 can be sequencing primers. One or more adapters 334 and 336 can be used for further amplification of adapter-labeled amplicons 338. One or more adapters 334 and 336 can be used for sequencing of adapter-labeled amplicons 338. Primer 342 can contain a plate index 344, allowing amplicons generated using the same set of barcodes or stochastic barcodes 310 to be sequenced in a single sequencing reaction using next-generation sequencing (NGS).

[0133] Barcoding the 5' end of nucleic acid targets The present disclosure includes systems, methods, compositions, and kits for attaching barcodes (e.g., stochastic barcodes) bearing molecular labels (or molecular indexes) to the 5' ends of barcoded or labeled nucleic acid targets (e.g., deoxyribonucleic acid molecules and ribonucleic acid molecules). The 5'-based transcript counting methods disclosed herein can complement or complement 3'-based transcript counting methods (e.g., the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ) and the Chromium™ Single Cell 3' Solution (10X Genomics, San Francisco, CA)). Barcoded nucleic acid targets can be used for sequence identification, transcript counting, alternative splicing analysis, mutation screening, and / or full-length sequencing in a high-throughput manner. Transcript counting at the 5' end (5' to the labeled target nucleic acid target) can reveal alternative splicing isoforms and variants at or near the 5' end of the nucleic acid molecule, including but not limited to splice variants, single nucleotide polymorphisms (SNPs), insertions, deletions, and substitutions. In some embodiments, the method can involve intramolecular hybridization.

[0134] 4A-4B show a schematic diagram of a non-limiting exemplary method 400 for gene-specifically labeling nucleic acid targets at their 5' ends. A barcode 420 (e.g., a stochastic barcode) containing a target-binding region (e.g., a poly(dT) tail 422) can bind to a polyadenylated RNA transcript 424, or other nucleic acid target, via a poly(dA) tail 426 for labeling or barcoding (e.g., unique labeling). The barcode 420 can include a molecular label 428 and a sample label 430 for labeling the transcript 424 and tracking the sample origin of the RNA transcript 424, respectively, along with one or more additional sequences (e.g., consensus sequences such as adapter sequences 432) flanking the molecular label (ML) 428 / sample label (SL) 430 region of each barcode 420 for subsequent reactions. The repertoire of molecular label sequences in barcodes for each sample can be sufficiently large for stochastic labeling of RNA transcripts.

[0135] After cDNA synthesis in block 402 to generate a barcoded cDNA molecule 434 containing the RNA transcript 424 (or a portion thereof), gene-specific methods can be used for 5' molecular barcoding. After gene-specific amplification in block 404, which can be selective, terminal transcriptase and deoxyadenosine triphosphate (dATP) can be added in block 406 to promote 3' poly(dA) tailing, generating an amplicon 436 containing a poly(A) tail 438. A short denaturation step in block 408 allows separation of the forward strand 436m and reverse strand 436c of the amplicon 436 (a barcoded cDNA molecule containing a poly(dA) tail). The reverse strand 436c of the amplicon 436 can hybridize intramolecularly via its poly(dA) tail 438 at the 3' end of the strand and the end of the poly(dT) region 422, forming a hairpin or stem-loop 440 in block 410. A polymerase (e.g., Klenow fragment) can then be used to replicate the barcode by extending from the poly(dA) tail 438 to form an extended barcoded reverse strand 442 in block 412. Gene-specific amplification in block 414 (e.g., optionally) can then be performed to amplify the gene of interest to generate an amplicon 444 comprising the barcode at its 5' end (relative to the RNA transcript 424) for sequencing in block 416. In some embodiments, method 400 includes one or both of gene-specific amplification of the barcoded cDNA molecule 434 in block 404 and gene-specific amplification of the extended barcoded reverse strand 442 in block 414.

[0136] 5A-5B show a schematic diagram of a non-limiting exemplary method 500 for labeling nucleic acid targets at the 5' end for whole-transcriptome analysis. A barcode 420 (e.g., a stochastic barcode) comprising a target binding region (e.g., a poly(dT) tail 422) can bind to a polyadenylated RNA transcript 424, or other nucleic acid target, via a poly(dA) tail 426 for labeling or barcoding (e.g., unique labeling). For example, the barcode 420 comprising the target binding region can bind to a nucleic acid target for labeling or barcoding. The barcode 420 can comprise a molecular label (ML) 428 and a sample label (SL) 430. The molecular label 428 and sample label 430 can be used, along with one or more additional sequences (e.g., consensus sequences such as adapter sequences 432) flanking the molecular label 428 / sample label 430 region of each barcode 420 for subsequent reactions, to label transcripts 424 or nucleic acid targets (e.g., antibody oligonucleotides associated with or dissociated from antibodies) and track the sample origin of transcripts 424, respectively. The repertoire of molecular label 428 sequences in barcodes for each sample can be sufficiently large for stochastic labeling of RNA transcripts 424 or nucleic acid targets.

[0137] After cDNA synthesis to generate barcoded cDNA molecules 434 in block 402, terminal transferase can be used to A-tail the 3' ends (equivalent to the 5' ends of the labeled RNA transcripts) of barcoded cDNA molecules 434 to generate cDNA molecules 436c each containing a 3' poly(dA) tail 438 in block 406. Intramolecular hybridization of cDNA molecules 436c with 3' poly(dA) tail 438 is initiated (e.g., by heating and cooling cycles or by diluting barcoded cDNA molecules 436c with poly(dA) tail 438), such that the new 3' poly(dA) tail 438 can anneal with poly(dT) tail 422 of the same labeled cDNA molecule to generate a hairpin or stem-loop structure 440 of the barcoded cDNA molecule in block 410. In block 412, a polymerase (e.g., Klenow enzyme) can be added with dNTPs to promote 3' extension beyond the new 3' poly(dA) tail 438, replicating the barcode (e.g., molecular label 428 present at the 5' end of the labeled cDNA molecule with stem-loop 440). In block 414, whole transcriptome amplification (WTA) can be performed using primers containing mirrored adapters 432, 432rc or sequences (or subsequences) of adapters 432, 432rc. In block 418, methods such as tagmentation or random priming can be used to generate smaller amplicon 444 fragments containing sequencing adapters (e.g., P5 446 and P7 448 sequences) for sequencing (e.g., using an Illumina (San Diego, CA, US) sequencer).In some embodiments, sequencing adapters for other sequencing methods or sequencers (e.g., sequencers from Pacific Biosciences of California, Inc. (Menlo Park, CA, US) or Oxford Nanopore Technologies Limited (Oxford, UK)) can be directly ligated to generate amplicons for sequencing.

[0138] The present disclosure includes methods for determining the number of nucleic acid targets in a sample. In some embodiments, the method includes contacting copies of a nucleic acid target 424 in a sample with a plurality of oligonucleotide barcodes 420, each of the plurality of oligonucleotide barcodes 420 comprising a molecular beacon sequence 428 and a target binding region (e.g., a poly(dT) sequence 422) capable of hybridizing to the nucleic acid target 424, wherein at least ten of the plurality of oligonucleotide barcodes 420 comprise different molecular beacon sequences 428; extending the copies of the nucleic acid target 424 hybridized to the oligonucleotide barcodes 420 to generate a plurality of nucleic acid molecules 434, in block 402, each of the plurality of nucleic acid molecules 434 comprising a sequence 450c complementary to at least a portion of the nucleic acid target 424; amplifying the plurality of barcoded nucleic acid molecules 434 to generate a plurality of amplified barcoded nucleic acid molecules 436, in block 404; and binding an oligonucleotide comprising a complement 438 of the target binding region 422 to the plurality of amplified barcoded nucleic acid molecules 436, in block 406. generating a plurality of barcoded nucleic acid molecules 436c, each comprising a target binding region 422 and a complement of the target binding region 438; hybridizing the target binding region 422 with the complement 438 of the target binding region 422 in each of the plurality of barcoded nucleic acid molecules 436c to form a stem-loop 440 in block 410; and extending the 3' ends of the plurality of barcoded nucleic acid molecules each comprising the stem-loop 440 in block 412 to form the stem-loop 440. to generate a plurality of extended barcoded nucleic acid molecules 442, each comprising a molecular label 428 and a complement 428rc of the molecular label; in block 414, amplifying the plurality of extended barcoded nucleic acid molecules 442 to generate a plurality of single-labeled nucleic acid molecules 444c, each comprising a complement 428rc of the molecular label; and determining the number of nucleic acid targets in the sample based on the number of complements 428rc of the molecular label having distinct sequences associated with the plurality of single-labeled nucleic acid molecules.

[0139] In some embodiments, molecular label 428 is hybridized to complement 428rc of molecular label after extending the 3' ends of the plurality of barcoded nucleic acid molecules with stem-loop 440. The method may include denaturing the plurality of extended barcoded nucleic acid molecules 442 to generate a plurality of single-labeled nucleic acid molecules 444c (which may be part of amplicons 444c) before amplifying the plurality of extended barcoded nucleic acid molecules 442. Contacting copies of nucleic acid targets 424 in the sample may include contacting the copies of the plurality of nucleic acid targets 424 with a plurality of oligonucleotide barcodes 420. Extending copies of nucleic acid targets 424 may include extending copies of the plurality of nucleic acid targets 424 hybridized to oligonucleotide barcodes 420 to generate a plurality of barcoded nucleic acid molecules 436c, each comprising a sequence 450c complementary to at least a portion of one of the plurality of nucleic acid targets 424. Determining the number of nucleic acid targets 424 may include determining the number of each of the plurality of nucleic acid targets 424 in the sample based on the number of complements 428rc of molecular labels having distinct sequences associated with single-labeled nucleic acid molecules among the plurality of single-labeled nucleic acid molecules 444c, each of which comprises a respective sequence 452c of the plurality of nucleic acid targets 424. The respective sequences 452c of the plurality of nucleic acid targets may comprise subsequences (including complements or reverse complements) of each of the plurality of nucleic acid targets 424.

[0140] The disclosure herein includes a method for determining the number of targets in a sample. In some embodiments, the method includes the steps of: barcoding (402) copies of a nucleic acid target 424 in the sample using a plurality of oligonucleotide barcodes 420 to generate a plurality of barcoded nucleic acid molecules 434, each of which includes a sequence 450c (e.g., a complementary sequence, a reverse complementary sequence, or a combination thereof) of the nucleic acid target 424, a molecular label 428, and a target binding region (e.g., a poly(dT) region 422), wherein at least 10 of the plurality of oligonucleotide barcodes 420 include different molecular label sequences 428; and binding (406) an oligonucleotide including a complement 438 of the target binding region 422 to the plurality of barcoded nucleic acid molecules 434, each of which includes a target binding region 422 and a target binding region 422. the target binding region 422 of each of the plurality of barcoded nucleic acid molecules 436c, hybridizing (410) the target binding region 422 with the complement 438 of the target binding region in each of the plurality of barcoded nucleic acid molecules 436c to form a stem-loop 440; extending (412) the 3' ends of the plurality of barcoded nucleic acid molecules to extend the stem-loop 440 and generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a molecular label 428 and a complement 428rc of the molecular label; and determining the number of nucleic acid targets 424 in the sample based on the number of complements 428rc of the molecular label having a distinct sequence associated with the plurality of extended barcoded nucleic acid molecules 442.

[0141] The present disclosure includes a method for binding oligonucleotide barcodes to targets in a sample. In some embodiments, the method includes the steps of: barcoding 402 copies of a nucleic acid target 424 in the sample using a plurality of oligonucleotide barcodes 420 to generate a plurality of barcoded nucleic acid molecules 434, each of which comprises a sequence 450c of the nucleic acid target 424, a molecular label 428, and a target binding region 422, wherein at least ten of the plurality of oligonucleotide barcodes 420 comprise different molecular label sequences 428; and binding an oligonucleotide comprising a complement 438 of the target binding region 422 to the plurality of barcoded nucleic acid molecules 434, each of which comprises a target binding region 422. 22 and a complement 438 of the target binding region 422, hybridizing (410) the target binding region 422 with the complement 438 of the target binding region 422 in each of the plurality of barcoded nucleic acid molecules 436c to form a stem-loop 440, and extending (412) the 3' ends of the plurality of barcoded nucleic acid molecules to extend the stem-loop 440 and generate a plurality of extended barcoded nucleic acid molecules 442, each of which comprises a molecular label 428 and a complement 428rc of the molecular label 428. In some embodiments, the method includes determining the number of nucleic acid targets 424 in the sample based on the number of molecular labels 428 having distinct sequences, their complements 428rc, or a combination thereof, associated with the plurality of extended barcoded nucleic acid molecules 442. For example, the number of nucleic acid targets 424 can be determined based on one or both of the molecular labels 428 having distinct sequences, their complements 428rc.

[0142] In some embodiments, barcoding (402) the copies of the plurality of targets 424 includes contacting the copies of the nucleic acid targets 424 with a plurality of oligonucleotide barcodes 420, each of the plurality of oligonucleotide barcodes 420 comprising a target binding region 422 capable of hybridizing to the nucleic acid targets 424, and extending (402) the copies of the nucleic acid targets 424 hybridized to the oligonucleotide barcodes 420 to generate a plurality of barcoded nucleic acid molecules 434.

[0143] In some embodiments, the method includes amplifying (404) a plurality of barcoded nucleic acid molecules 434 to generate a plurality of amplified barcoded nucleic acid molecules 436c, wherein binding an oligonucleotide comprising a complement 438 of the target binding region 422 includes binding an oligonucleotide comprising a complement 438 of the target binding region to the plurality of amplified barcoded nucleic acid molecules to generate a plurality of barcoded nucleic acid molecules 436r, each comprising a target binding region 422 and a complement 438 of the target binding region.

[0144] Gene-specific analysis. In some embodiments, the method (e.g., method 400) includes amplifying (414) a plurality of extended barcoded nucleic acid molecules 442 to generate a plurality of single-labeled nucleic acid molecules 444c, each comprising a complement 428rc of a molecular label 428. The single-labeled nucleic acid molecules 444c can be generated when the amplicon 444 containing them is denatured. Determining the number of nucleic acid targets 424 in the sample can include determining the number of nucleic acid targets 424 in the sample based on the number of complements 428rc of molecular labels 428 having distinct sequences associated with the plurality of single-labeled nucleic acid molecules 444c.

[0145] Whole transcriptome analysis. In some embodiments, the method (e.g., method 500) includes amplifying (414) a plurality of extended barcoded nucleic acid molecules 442 to generate a plurality of extended barcoded nucleic acid molecule copies 444c. Determining the number of nucleic acid targets 424 in the sample includes determining the number of nucleic acid targets 424 in the sample based on the number of complements 428rc of molecular labels 428 having distinct sequences associated with the plurality of extended barcoded nucleic acid molecule copies 444c. The plurality of extended barcoded nucleic acid molecule copies 444c can be formed when the amplicons 444 containing them are denatured. In some embodiments, the sequence of the nucleic acid target in the plurality of barcoded nucleic acid molecules includes a subsequence 452c of the nucleic acid target. The target binding region may include a gene-specific sequence. Binding (406) an oligonucleotide comprising a complement 438 of the target binding region 422 may include ligating an oligonucleotide comprising a complement 438 of the target binding region 422 to the plurality of barcoded nucleic acid molecules 434. In some embodiments, the target binding region may comprise a poly(dT) sequence 422. Binding an oligonucleotide comprising a complement 438 of the target binding region 422 comprises adding a plurality of adenosine monophosphates to the plurality of barcoded nucleic acid molecules 434 using terminal deoxynucleotidyl transferase.

[0146] In some embodiments, extending the copy of the nucleic acid target 424 hybridized to the oligonucleotide barcode 420 may include reverse transcribing the copy of the nucleic acid target 424 hybridized to the oligonucleotide barcode 420 to generate a plurality of barcoded complementary deoxyribonucleic acid (cDNA) molecules 434. Extending the copy of the nucleic acid target 424 hybridized to the oligonucleotide barcode 420 may include extending (402) the copy of the nucleic acid target 424 hybridized to the oligonucleotide barcode 420 using a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. The DNA polymerase may comprise a Klenow fragment. In some embodiments, the method includes obtaining sequence information of the plurality of extended barcoded nucleic acid molecules 442. Obtaining the sequence information may include attaching sequencing adaptors (e.g., P5 446 and P7 448 adaptors) to the plurality of extended barcoded nucleic acid molecules 442. In some embodiments, the complement of the target binding region 438 may comprise the reverse complement of the target binding region. The complement of the target binding region 438 may comprise the complementary sequence of the target binding region. The complement of the molecular label 428rc may comprise the reverse complement of the molecular label. The complement of the molecular label may comprise the complementary sequence of the molecular label.

[0147] In some embodiments, the plurality of barcoded nucleic acid molecules 434 may comprise barcoded deoxyribonucleic acid (DNA) molecules. The barcoded nucleic acid molecules 434 may comprise barcoded ribonucleic acid (RNA) molecules. The nucleic acid target 424 may comprise a nucleic acid molecule. The nucleic acid molecule may comprise ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof.

[0148] Antibody oligonucleotide. In some embodiments, the nucleic acid target may include a cellular component binding reagent. Cell-binding reagents associated with nucleic acid targets (e.g., antibody oligonucleotides, such as sample-indexing oligonucleotides) are described in US2018 / 0088112 and U.S. Application No. 15 / 937,713, filed March 27, 2018, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the 5' barcoding method of the present disclosure can be used to obtain multi-omics information of single cells, such as genomics, chromatin accessibility, methylomics, transcriptomics, and proteomics. The nucleic acid molecule may be associated with a cellular component binding reagent. The method may include a step of dissociating the nucleic acid molecule from the cellular component binding reagent. In some embodiments, each molecular label 428 of the plurality of oligonucleotide barcodes 420 comprises at least six nucleotides. The oligonucleotide barcodes 420 may comprise the same sample label 430. Each sample label 430 of the plurality of oligonucleotide barcodes 420 may comprise at least six nucleotides. The oligonucleotide barcodes 420 may comprise the same cell label. Each cell label of the plurality of oligonucleotide barcodes 420 may comprise at least six nucleotides.

[0149] In some embodiments, at least one of the plurality of barcoded nucleic acid molecules 436c is associated with a solid support when, in each of the plurality of barcoded nucleic acid molecules, the target binding region hybridizes (410) to a complement of the target binding region to form a stem-loop. At least one of the plurality of barcoded nucleic acid molecules 436c may dissociate from the solid support when, in each of the plurality of barcoded nucleic acid molecules 436c, the target binding region 422 hybridizes (410) to a complement 438 of the target binding region 422 to form a stem-loop 440. At least one of the plurality of barcoded nucleic acid molecules 436c may be associated with a solid support when, in each of the plurality of barcoded nucleic acid molecules 436c, the target binding region 422 hybridizes (410) to a complement 438 of the target binding region to form a stem-loop 440.

[0150] In some embodiments, at least one of the plurality of barcoded nucleic acid molecules is associated with the solid support when the 3' ends of the plurality of barcoded nucleic acid molecules are extended (412) to extend the stem-loop 440 and generate a plurality of extended barcoded nucleic acid molecules 442, each comprising a molecular label 428 and a complement 428rc of the molecular label. At least one of the plurality of barcoded nucleic acid molecules may be dissociated from the solid support when the 3' ends of the plurality of barcoded nucleic acid molecules are extended (412) to extend the stem-loop 440 and generate a plurality of extended barcoded nucleic acid molecules 442, each comprising a molecular label 428 and a complement 428rc of the molecular label. At least one of the plurality of barcoded nucleic acid molecules 436c may be associated with a solid support when the 3' end of the plurality of barcoded nucleic acid molecules is extended (412) to extend stem-loop 440 and generate a plurality of extended barcoded nucleic acid molecules 442, each comprising a molecular label 428 and a complement 428rc of the molecular label. The solid support may comprise a synthetic particle 454. The solid support may comprise a planar or substantially planar surface (e.g., a slide such as a microscope slide or a coverslip).

[0151] In some embodiments, at least one of the plurality of barcoded nucleic acid molecules 436c is in solution when the target binding region 422 and the complement 438 of the target binding region 422 in each of the plurality of barcoded nucleic acid molecules 436c hybridize (410) to form a stem-loop 440. For example, such intramolecular hybridization can occur when the concentration of the plurality of barcoded nucleic acid molecules 436c in solution is sufficiently low. At least one of the plurality of barcoded nucleic acid molecules can be in solution when the 3' ends of the plurality of barcoded nucleic acid molecules are extended (412) to extend the stem-loop 440 and generate a plurality of extended barcoded nucleic acid molecules 442, each including a molecular label 428 and a complement 428rc of the molecular label.

[0152] In some embodiments, the sample includes a single cell, and the method includes associating a synthetic particle 454 including a plurality of oligonucleotide barcodes 420 with the single cell in the sample. The method may include lysing the single cell after associating the synthetic particle 454 with the single cell. Lysing the single cell may include heating the sample, contacting the sample with a surfactant, altering the pH of the sample, or any combination thereof. The synthetic particle and the single cell may be in the same well. The synthetic particle and the single cell may be in the same droplet.

[0153] In some embodiments, at least one of the plurality of oligonucleotide barcodes 420 may be immobilized on a synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be partially immobilized on a synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be encapsulated within a synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be partially encapsulated within a synthetic particle 454. The synthetic particle 454 may be disintegrable. The synthetic particle 454 may comprise beads. The beads 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 synthetic particle 454 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 synthetic particle 454 may comprise a collapsible hydrogel particle. Each of the plurality of oligonucleotide barcodes 420 may comprise a linker functional group. The synthetic particle 454 may comprise a solid support functional group. The support functional group and the linker functional group may be associated with each other. The linker functional group and the support functional group may be independently selected from the group consisting of C6, biotin, streptavidin, primary amine, aldehyde, ketone, and any combination thereof.

[0154] Kit for barcoding at the 5' end of nucleic acid targets The disclosure herein includes kits for binding oligonucleotide barcodes 420 to targets 424 in a sample, determining the number of targets 424 in a sample, and / or determining the number of nucleic acid targets 424 in a sample. In some embodiments, the kits include a plurality of oligonucleotide barcodes 420, each of which includes a molecular label 428 and a target binding region (e.g., a poly(dT) sequence 422), and at least 10 of the plurality of oligonucleotide barcodes 420 include different molecular label sequences 428; a terminal deoxynucleotidyl transferase or ligase; and a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. The DNA polymerase may include a Klenow fragment. The kit may include a buffer. The kit may include a cartridge. The kit may include one or more reagents for a reverse transcription reaction. The kit may include one or more reagents for an amplification reaction. In some embodiments, the target binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. The oligonucleotide barcodes may comprise the same sample label and / or the same cell label. Each sample label and / or cell label of the plurality of oligonucleotide barcodes may comprise at least six nucleotides. Each molecular label of the plurality of oligonucleotide barcodes may comprise at least six nucleotides.

[0155] In some embodiments, at least one of the plurality of oligonucleotide barcodes 420 is partially immobilized on a synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be partially immobilized on a synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be encapsulated within a synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be partially encapsulated within a synthetic particle 454. The synthetic particle 454 may be disintegrable. The synthetic particle 454 may comprise beads. The beads 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 synthetic 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 synthetic particles 454 may comprise collapsible hydrogel particles. Each of the plurality of oligonucleotide barcodes may comprise a linker functional group. The synthetic particles 454 may comprise a solid support functional group. The support functional group and the linker functional group may be associated with each other. The linker functional group and the support functional group may be independently selected from the group consisting of C6, biotin, streptavidin, primary amine, aldehyde, ketone, and any combination thereof.

[0156] Determination of 5' transcript sequence High-throughput single-cell RNA sequencing has transformed our understanding of complex and heterogeneous biological samples. However, most methods only allow for 3'-only analysis of mRNA transcript information, which can limit analysis of highly variable loci due to rearrangements such as splice variants, alternative transcription start sites, and VDJ junctions between T-cell and B-cell receptors and antibodies. As disclosed herein, mRNA molecules were captured and sequencing libraries were generated for both the 3' and 5' ends of transcripts in a high-throughput manner using the BD Rhapsody platform.

[0157] The method of the present disclosure can be used to identify the VDJ regions of B cell receptors (BCRs), T cell receptors (TCRs), and antibodies. VDJ recombination, also known as somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) (e.g., BCR) and T cell receptor (TCR) production in the immune system. VDJ recombination allows variable (V), diversity (D), and joining (J) gene segments to be combined in a nearly random manner. This randomness in the selection of various genes allows for the encoding of a variety of proteins that match antigens derived from bacteria, viruses, parasites, dysfunctional cells such as tumor cells, and pollen.

[0158] The VDJ region can contain a large 3 Mb locus containing variable (V), diversity (D), and joining (J) genes. These are the segments that can participate in VDJ recombination. There may also be constant genes that may not undergo VDJ recombination. The initial event in VDJ recombination at this locus may be the rearrangement of one of the D genes with one of the J genes. After this, one of the V genes can be added to this DJ rearrangement to form a functional VDJ rearranged gene that subsequently encodes the variable segment of a heavy chain protein. Both of these steps can be catalyzed by recombinase enzymes that can delete intervening DNA. This recombination process occurs stepwise in precursor B cells, providing the diversity needed for the antibody repertoire. Each B cell can produce only one antibody (e.g., BCR). This specificity can be achieved by allelic exclusion, where functional rearrangement of one allele signals the prevention of further recombination of the second allele. In some embodiments, the sample comprises immune cells, which may include, for example, T cells, B cells, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, B cell precursors, T cell precursors, natural killer cells, Tc cells, Th cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells and / or macrophages, or any combination thereof.

[0159] T cells may be derived from a single T cell or a T cell clone, which can refer to T cells with the same TCR. T cells may also be part of a T cell line, which can include a mixed population of T cell clones and T cells with different TCRs, all of which can recognize the same target (e.g., antigen, tumor, virus). T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and tumors. T cells can be obtained from a unit of blood collected from a subject, such as using Ficoll separation. Cells derived from an individual's circulating blood can be obtained by apheresis or leukapheresis. The apheresis product can include T cells, monocytes, granulocytes, lymphocytes, including B cells, other nucleated leukocytes, red blood cells, and platelets. The cells can be washed and resuspended in medium to isolate the cells of interest.

[0160] T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4, CD4+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T or XCYTE DYNABEADS™, for a period of time sufficient for positive selection of the desired T cells. Immune cells (e.g., T cells and B cells) can be antigen-specific (e.g., tumor-specific). In some embodiments, the cell may be an antigen-presenting cell (APC), such as a B cell, an activated B cell from a lymph node, a lymphoblastoid cell, a resting B cell, or a neoplastic B cell from, for example, a lymphoma. APC may refer to a B cell or a follicular dendritic cell that expresses at least one of the BCRC proteins on its surface.

[0161] The disclosed methods can be used to track the molecular phenotype of single T cells. Various T cell subtypes can be distinguished by the expression of various molecular markers. T cells express unique T cell receptors (TCRs) from a diverse repertoire of TCRs. In most T cells, the TCR may be composed of a heterodimer of α and β chains, and each functional chain may be the product of somatic DNA recombination events during T cell development, allowing the expression of over one million different TCRs in a single individual. TCRs can be used to define the identity of individual T cells and enable lineage tracing of T cell clonal expansion during an immune response. The disclosed immunological methods can be used in a variety of ways, including, but not limited to, identifying unique TCR α and TCR β chain pairings in single T cells, quantifying TCR and marker expression at the single-cell level, identifying TCR diversity in individuals, characterizing the TCR repertoire expressed in different T cell populations, determining the functionality of TCR alpha and beta chain alleles, and identifying clonal expansion of T cells during an immune response.

[0162] T cell receptor chain pairing T cell receptors (TCRs) are recognition molecules present on the surface of T lymphocytes. T cell receptors found on the surface of T cells can be composed of two glycoprotein subunits, called alpha and beta chains. Both chains contain a molecular weight of approximately 40 kDa and can have variable and constant domains. Genes encoding the alpha and beta chains can be organized into libraries of V, D, and J regions, where genes are formed by gene rearrangement. TCRs can recognize antigens presented by antigen-presenting cells as part of a complex with specific self-molecules encoded by histocompatibility genes. The most dominant histocompatibility genes are known as major histocompatibility complexes (MHC). Thus, the complex recognized by the T cell receptor consists of an MHC / peptide ligand.

[0163] In some embodiments, the disclosed methods, devices, and systems can be used for sequencing and pairing T cell receptors. The disclosed methods, devices, and systems can be used to sequence T cell receptor alpha and beta chains, pair alpha and beta chains, and / or determine functional copies of T cell receptor alpha chains. A single cell can be contained in a single compartment (e.g., a well) containing a single solid support (e.g., a bead). The cell can be lysed. The bead can contain a stochastic label that can bind to a specific position within the TCR alpha and / or beta chain. The TCR alpha and beta molecules associated with the solid support can be subjected to the disclosed molecular biology methods, including reverse transcription, amplification, and sequencing. TCR alpha and beta chains containing the same cell label are considered to be derived from the same single cell, thereby allowing the TCR alpha and beta chains to be paired.

[0164] Heavy and light chain pairing in the antibody repertoire The disclosed methods, devices, and systems can be used to pair BCR receptors and antibody heavy and light chains. The disclosed methods allow for the determination of immune receptor and antibody repertoires in individual organisms or cell populations. The disclosed methods can help determine the pairs of polypeptide chains that make up immune receptors. B cells and T cells each express immune receptors, with B cells expressing immunoglobulins and BCRs, and T cells expressing T cell receptors (TCRs). Both types of immune receptors can contain two polypeptide chains. Immunoglobulins can contain a variable heavy (VH) chain and a variable light (VL) chain. There are two types of TCRs: one consisting of an alpha chain and a beta chain, and one consisting of a delta chain and a gamma chain. The polypeptides in immune receptors can contain a constant region and a variable region. The variable region can arise from recombination and end-joining rearrangement of gene fragments in the chromosomes of B cells or T cells. In B cells, further diversification of the variable region can occur through somatic hypermutation. The immune system has a large repertoire of receptors, and any given pair of receptors expressed by a lymphocyte may be encoded by a separate, unique pair of transcripts. Knowledge of the sequences of pairs of immune receptor chains expressed in a single cell can be used to ascertain the immune repertoire of a given individual or cell population.

[0165] In some embodiments, the disclosed method, device, and system can be used for antibody sequencing and pairing. The disclosed method, device, and system can be used for antibody heavy and light chain sequencing (e.g., in B cells) and / or heavy and light chain pairing. A single cell can be contained in a single compartment (e.g., well) containing a single solid support (e.g., bead). The cell can be lysed. The beads may contain stochastic labels that can bind to specific positions within the heavy and / or light chains of antibodies (e.g., in B cells). The heavy and light chain molecules associated with the solid support can be subjected to the molecular biology methods of the present disclosure, including reverse transcription, amplification, and sequencing. The heavy and light chains of antibodies containing the same cell label are considered to be derived from the same single cell, thereby allowing the heavy and light chains of antibodies to be paired.

[0166] In some embodiments, a method for labeling a nucleic acid target in a sample is provided. In some embodiments, the method includes contacting a copy of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copy of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each comprising a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; hybridizing the complement of the target binding region of each barcoded nucleic acid molecule to (i) an oligonucleotide barcode of the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules; and extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each comprising the first molecular label and the second molecular label. The method may include determining the copy number of a nucleic acid target in a sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with a plurality of extended barcoded nucleic acid molecules, or products thereof.

[0167] In some embodiments, a method for determining the number of nucleic acid targets in a sample is provided, the method comprising: contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; and (i) combining the complement of the target binding region of each barcoded nucleic acid molecule with a plurality of oligonucleotide barcodes. the method includes hybridizing (i) an oligonucleotide barcode among the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules; extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a first molecular label and a second molecular label; and determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with the plurality of extended barcoded nucleic acid molecules, or products thereof.

[0168] In some embodiments, a method for generating and analyzing single-labeled nucleic acid molecules is provided.The method may include: amplifying a plurality of elongated barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules, each of which comprises a first molecular label or a second molecular label; and determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels with distinct sequences associated with the plurality of single-labeled nucleic acid molecules.In some embodiments, determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels with distinct sequences associated with the plurality of single-labeled nucleic acid molecules. The method may include amplifying a plurality of extended barcoded nucleic acid molecules to produce a plurality of copies of the extended barcoded nucleic acid molecules, wherein determining the copy number of the nucleic acid target in the sample includes determining the copy number of the nucleic acid target in the sample based on (i) the number of first molecular labels having distinct sequences associated with the copies of the plurality of extended barcoded nucleic acid molecules, or products thereof, and / or (ii) the number of second molecular labels having distinct sequences associated with the copies of the plurality of extended barcoded nucleic acid molecules, or products thereof.

[0169] Also provided herein are methods, systems, compositions, and kits for determining the number of nucleic acid targets in a sample. In some embodiments, the methods include contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target in the presence of a template switch oligonucleotide comprising a reverse transcriptase and a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; and interfering the complement of the target binding region of each barcoded nucleic acid molecule with (i) the plurality of oligonucleotide barcodes. the method further comprises hybridizing (i) an oligonucleotide barcode among the plurality of barcoded nucleic acid molecules, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules; extending the 3' ends of the plurality of barcoded nucleic acid molecules to produce a plurality of extended barcoded nucleic acid molecules, each of which comprises the first molecular label and the second molecular label; amplifying the plurality of extended barcoded nucleic acid molecules to produce a plurality of single-labeled nucleic acid molecules, each of which comprises the first molecular label or the second molecular label; and determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels having distinct sequences associated with the plurality of single-labeled nucleic acid molecules.

[0170] Some embodiments of the methods provided herein include determining the copy number of a nucleic acid target in a sample based on the number of first molecular labels with distinct sequences associated with a plurality of single-labeled nucleic acid molecules. In some embodiments, the method includes denaturing the plurality of barcoded nucleic acid molecules before hybridizing the complement of the target binding region of each barcoded nucleic acid molecule with (i) an oligonucleotide barcode among the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) the target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules. The method may include denaturing the plurality of extended barcoded nucleic acid molecules before amplifying the plurality of extended barcoded nucleic acid molecules. Determining the copy number of the nucleic acid target may include determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of second molecular labels with distinct sequences associated with a single-labeled nucleic acid molecule among the plurality of single-labeled nucleic acid molecules containing the respective sequences of the plurality of nucleic acid targets. Determining the copy number of the nucleic acid target may include determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of first molecular labels having distinct sequences associated with single-labeled nucleic acid molecules among a plurality of single-labeled nucleic acid molecules containing the respective sequences of the plurality of nucleic acid targets. Each sequence of the plurality of nucleic acid targets may include a subsequence of each of the plurality of nucleic acid targets. The sequence of the nucleic acid target in the plurality of barcoded nucleic acid molecules may include a subsequence of the nucleic acid target.

[0171] In some embodiments, the method involves adding (e.g., by a template switching reaction) the complement of the target binding region to the end (e.g., the 3' end) of the barcoded nucleic acid molecule. In some embodiments, the method involves i) intramolecular hybridization and / or ii) intermolecular hybridization of the target binding region of the oligonucleotide barcode (or its product, such as another barcoded nucleic acid molecule or its amplicon), followed by extension to generate an extended barcoded nucleic acid molecule. The extended barcoded nucleic acid molecule may be barcoded at both the 3' and 5' ends. In some embodiments, intramolecular hybridization of the barcoded molecule forms a hairpin loop containing the captured mRNA transcript on a 3' poly(dT) capture bead. The mRNA molecule may be captured on the bead by a poly(A) tail attached to the target binding region of the oligonucleotide barcode. After hybridization, template switching can be used to attach a poly(dA) tail to the 5' end of the captured transcript. The new poly(dA) tail can then hybridize and release a capture oligonucleotide (e.g., a barcode, such as a stochastic barcode) on the same bead. After extension, the mRNA molecule can be barcoded at both the 3' and 5' ends. This allows for the generation of 3'- and 5'-barcoded transcripts that can be sequenced, for example, on Illumina sequencing platforms. Access to the barcoded 5' sequence can enable the detection of splice variants and sequence variations that occur in the variable regions of T cell receptors (TCRs) and B cell receptors (BCRs), as well as the 5' end of the transcript.

[0172] Figures 6A-6K show schematic diagrams of non-limiting, exemplary workflows for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' and / or 3' barcoding. BD® Rhapsody™ beads are barcoded solid beads whose integrity is maintained through a wide range of physical and chemical manipulations. After poly(A) capture of mRNA on the beads, reverse transcription and template switching can be performed to add a poly(dA) tail to the 3' end of the barcoded cDNA. The added poly(dA) tail allows the bead-bound cDNA to self-hybridize to the oligo(dT) region of a barcode (e.g., a stochastic barcode) on the same bead, forming a bridge-loop structure. Klenow extension of the bridge-loop can generate new barcoded cDNA molecules derived from the same mRNA transcript with the first barcoded cDNA in the opposite orientation, allowing both the 3' and 5' ends to be linked to molecular barcodes.

[0173] The methods disclosed herein can enable 3'- and / or 5'-based sequencing. This method can provide flexibility in sequencing. In some embodiments, the methods can enable profiling of both T and B cell immune repertoires in the Rhapsody™ system for samples, e.g., mouse and human samples, without changing the protocol or product conformation apart from the primers used. In some embodiments, 3' and / or 5' gene expression profiling of V(D)J can be performed. In some embodiments, both phenotypic markers and V(D)J sequences of T and B cells in a single-cell platform can be investigated. In some embodiments, both 3' and 5' information of these transcripts can be captured in a single experiment. The methods disclosed herein can enable V(D)J detection (e.g., hypermutation) of both T and B cells.

[0174] The methods and systems described herein can be used in conjunction with methods and systems that use antibodies associated with (e.g., bound to or conjugated to) oligonucleotides (also referred to herein as AbOs or AbOligos). Embodiments using AbOs to determine protein expression profiles in single cells and track sample origin are described in U.S. Patent Application No. 15 / 715,028, published as U.S. Patent Application Publication No. 2018 / 0088112, and U.S. Patent Application No. 15 / 937,713, the contents of each of which are incorporated herein by reference in their entireties. In some embodiments, the methods disclosed herein enable T and B cell V(D)J profiling, 3' targeting, 5' targeting, 3' whole transcriptome amplification (WTA), 5' WTA, protein expression profiling using AbOs, and / or sample multiplexing in a single experiment. FIG. 7 shows non-limiting exemplary diagrams for implementing the V(D)J workflow, the antibody-oligonucleotide (AbO) workflow, and the single-cell mRNA expression profile workflow (e.g., the BD Rhapsody targeted workflow).

[0175] Template switching reaction 6A-6K show schematic diagrams of non-limiting exemplary workflows for determining the sequence of a nucleic acid target (e.g., the V(D)J region of an immune receptor) using 5' barcoding and / or 3' barcoding. The barcode (e.g., a stochastic barcode, oligonucleotide barcode 602) may include a target binding region (e.g., poly(dT) 604) that can bind to a nucleic acid target (e.g., a polyadenylated RNA transcript 606) or to other nucleic acid targets via a poly(dA) tail 608 for labeling or barcoding (e.g., unique labeling). The target binding region may include a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. In some embodiments, the barcode is associated with a solid support (e.g., particle 610). Multiple barcodes 602 may be associated with particle 610. In some embodiments, the particle is a bead. The beads can be polymer beads, such as deformable beads or gel beads (e.g., gel beads from 10X Genomics (San Francisco, CA)) functionalized with barcodes or stochastic barcodes. In some implementations, the gel beads can comprise a polymer-based gel. Gel beads can be generated, for example, by encapsulating one or more polymer precursors in droplets. Gel beads can be generated when the polymer precursors are exposed to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0176] 6A shows a non-limiting exemplary embodiment of reverse transcription reaction 600a. During reverse transcription 600a, upon reaching the end of oligonucleotide barcode 602, the activity of the terminal transferase of an enzyme (e.g., a reverse transcriptase such as Moloney Murine Leukemia Virus (MMLV)) adds several additional nucleotides (e.g., deoxycytidine, CCC 612) to the 3' end of newly synthesized cDNA sequence strand 614c (the antisense sequence of RNA sequence 614r). These CCC bases 612 can serve as anchor sites for template switch oligonucleotide 616 (e.g., a template switching oligonucleotide) containing a sequence complementary to the tailed sequence (e.g., rGrGrG 618). Template switch oligonucleotide 616 can include at least a portion of target binding region 604. Upon base pairing between rGrGrG 618 and the added deoxycytidine stretch 612, the enzyme "switches" the template strand from oligonucleotide barcode 602 to template switch oligonucleotide 616 and continues replication toward the 5' end of template switch oligonucleotide 616. Thus, the resulting first-strand labeled cDNA (e.g., barcoded nucleic acid molecule 620) contains the reverse complement sequence of template switch oligonucleotide 616 and thus may include the complement (e.g., reverse complement) of the target binding region (e.g., poly(dA) 608). Barcoded nucleic acid molecule 620 may include cDNA 614c (the reverse complement sequence of RNA sequence 614r). The reaction can be performed in the presence of one or more additives configured to reduce secondary structure (e.g., ethylene glycol). Barcoded nucleic acid molecule 620 may also include several labels.Oligonucleotide barcodes 602 may include a first molecular label (ML1) 622 and a sample label (e.g., a compartment label, cell label (CL) 624) for labeling transcripts 606 and tracking the sample origin of RNA transcripts 606 (or nucleic acid targets, such as antibody oligonucleotides, associated with or dissociated from antibodies), along with one or more additional sequences, such as a first universal sequence 626 (e.g., a lead 1 sequence), adjacent to the first molecular label 622 / cell label 624 region of each barcode 602 for subsequent reactions. The repertoire of sequences of molecular labels in oligonucleotide barcodes for each sample may be sufficiently large for stochastic labeling of RNA transcripts. In some embodiments, the sample label is a compartment label. In some embodiments, the sample label is a cell label. Barcoded nucleic acid molecule 620 may undergo a denaturing step 600b (e.g., a denaturing step), thereby producing a single-stranded barcoded nucleic acid molecule 621.

[0177] In some embodiments, the first molecular label is hybridized to the second molecular label after extending the 3'-end of a plurality of barcoded nucleic acid molecules. In some embodiments, each extended barcoded nucleic acid molecule comprises a first molecular label, a second molecular label, a target binding region, and a complement of the target binding region. In some embodiments, the complement of the target binding region is complementary to a portion of the target binding region. In some embodiments, the target binding region comprises a gene-specific sequence. In some embodiments, the target binding region comprises a poly(dT) sequence.

[0178] The term "template switching" can refer to the ability of reverse transcriptase to switch from an initial nucleic acid sequence template to the 3' end of a new nucleic acid sequence template that has little or no complementarity to the 3' end of the nucleic acid synthesized from the initial template. An example of template switching is the ability of reverse transcriptase to switch from the initial nucleic acid sequence template / primer substrate to the 3' end of a new nucleic acid sequence template that has little or no complementarity to the 3' end of the nucleic acid primer strand. Template switching, for example, allows DNA copies to be prepared using reverse transcriptase to switch from the initial nucleic acid sequence template to the 3' end of a new nucleic acid sequence template that has little or no complementarity to the 3' end of the DNA synthesized from the initial template, thereby enabling the synthesis of successive product DNAs in which adapter sequences are directly linked to target oligonucleotide sequences without ligation. Template switching can involve adapter ligation, homopolymer tailing (e.g., polyadenylation), random primers, or oligonucleotides that polymerases can associate with. In any of the above-described embodiments, template switching may be used to introduce the target binding region or its complement.

[0179] In some embodiments, the reverse transcriptase is capable of terminal transferase activity. In some embodiments, the template switch oligonucleotide comprises one or more 3' ribonucleotides. In some embodiments, the template switch oligonucleotide comprises three 3' ribonucleotides. In some embodiments, the 3' ribonucleotide comprises a guanine. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase. In some embodiments, the viral reverse transcriptase is a Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the template switching oligonucleotide comprises SEQ ID NO: 1.

[0180] The complement of a target binding region may comprise the reverse complement of the target binding region or the complementary sequence of the target binding region. The complement of a molecular label may comprise the reverse complement of the molecular label or the complementary sequence of the molecular label. In some embodiments, the plurality of barcoded nucleic acid molecules may comprise barcoded deoxyribonucleic acid (DNA) molecules and / or barcoded ribonucleic acid (RNA) molecules. In some embodiments, the nucleic acid target comprises a nucleic acid molecule (e.g., ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof). In some embodiments, the mRNA encodes an immune receptor. The nucleic acid target may comprise a cellular component binding reagent. In some embodiments, the nucleic acid molecule is associated with a cellular component binding reagent. The method may include dissociating the nucleic acid molecule from the cellular component binding reagent. In some embodiments, at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences. Each molecular label of the plurality of oligonucleotide barcodes may comprise at least six nucleotides.

[0181] In some embodiments, multiple oligonucleotide barcodes are associated with a solid support. Multiple oligonucleotide barcodes associated with the same solid support may each include the same sample label. Each sample label of the multiple oligonucleotide barcodes may include at least six nucleotides. Each multiple oligonucleotide barcode may include a cell label. Each cell label of the multiple oligonucleotide barcodes may include at least six nucleotides. Oligonucleotide barcodes associated with the same solid support may include the same cell label. Oligonucleotide barcodes associated with different solid supports may include different cell labels. Each multiple extended barcoded nucleic acid molecule may include a cell label and a complement of the cell label. The complement of the cell label may include the reverse complement sequence of the cell label or the complementary sequence of the cell label. The method may include extending a plurality of oligonucleotide barcodes hybridized to copies of a nucleic acid target in the presence of one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof. In some embodiments, the solid support may comprise a synthetic particle. In some embodiments, the solid support may comprise a planar surface.

[0182] The sample may include a single cell, and the method may include associating synthetic particles comprising a plurality of oligonucleotide barcodes with the single cell in the sample. The method may include lysing the single cell after associating the synthetic particles with the single cell. Lysing the single cell may include heating the sample, contacting the sample with a surfactant, changing the pH of the sample, or any combination thereof. In some embodiments, the synthetic particles and the single cell are in the same well. In some embodiments, the synthetic particles and the single cell are in the same droplet. In some embodiments, at least one of the plurality of oligonucleotide barcodes is immobilized on the synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially immobilized on the synthetic particle. At least one of the plurality of oligonucleotide barcodes may be encapsulated within the synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially encapsulated within the synthetic particle. In some embodiments, the synthetic particle is disintegratable. The synthetic particle may include a bead. The beads 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 synthetic 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. In some embodiments, the synthetic particles may include disintegrable hydrogel particles.Each of the plurality of oligonucleotide barcodes may include a linker functional group, the synthetic particle may include a solid support functional group, and / or the support functional group and the linker functional group may be associated with each other. In some embodiments, the linker functional group and the support functional group are independently selected from the group consisting of C6, biotin, streptavidin, primary amines, aldehydes, ketones, and any combination thereof.

[0183] Intramolecular hybridization of barcoded nucleic acid molecules In some embodiments, hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with its own target binding region comprises intramolecular hybridization of the target binding region in the barcoded nucleic acid molecule with the complement of the target binding region, forming a stem-loop. In some embodiments, the second molecular label is the complement of the first molecular label.

[0184] The workflow may include intramolecular hybridization of single-stranded barcoded nucleic acid molecule 621, as shown in the non-limiting exemplary schematic diagram of FIG. 6B. The workflow may include intramolecular hybridization 600c1 of the complement of target binding region 604 and target binding region 608 within single-stranded barcoded nucleic acid molecule 621 to form a stem-loop. The workflow may include extending (600c2) the 3' end of the stem-loop of single-stranded barcoded nucleic acid molecule 621 to generate extended barcoded nucleic acid molecule 620c. Extended barcoded nucleic acid molecule 620c may include the complement (e.g., reverse complement) of first molecular label 622rc, the complement (e.g., reverse complement) of cell label 624rc, and / or the complement (e.g., reverse complement) of first universal sequence 626rc. The workflow may include denaturing (600c3) the extended barcoded nucleic acid molecule 620c to generate a single-stranded extended barcoded nucleic acid molecule 620cd. In some embodiments, intermolecular hybridization 600c1 and / or extension 600c2 are performed in the presence of a high salt buffer and / or PEG. In some embodiments, extension is performed using a DNA polymerase that lacks at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity (e.g., Klenow fragment).

[0185] The single-stranded, extended barcoded nucleic acid molecule 620cd may include barcodes (e.g., cellular and molecular labels) at both the 5' and 3' ends of the target nucleic acid molecule (e.g., transcript), thereby enabling more extensive analysis of the target nucleic acid molecule for sequence identification, transcript counting, alternative splicing analysis, mutation screening, and / or full-length sequencing compared to analysis of a target nucleic acid molecule having only one barcode at one end. The single-stranded, extended barcoded nucleic acid molecule 620cd may serve as a template for one or more amplification reactions (e.g., PCR), such as, for example, the non-limiting exemplary amplification schemes shown in Figures 6C-6D. Amplification may include target-specific (e.g., gene-specific) cDNA amplification. For example, single-stranded, extended barcoded nucleic acid molecule 620cd may undergo a first round of amplification ("PCR1") 600c4 using a universal oligonucleotide primer 646 comprising the sequence of a first universal sequence (or its complement) and a target-specific primer (e.g., target-specific primer 648 and / or target-specific primer 650). PCR1 600c4 may include amplifying a 5' region of single-stranded, extended barcoded nucleic acid molecule 620cd comprising universal oligonucleotide primer 646 and target-specific primer 648, thereby generating single-labeled nucleic acid molecule 620c1 comprising first molecular label 622, cell label 624, first universal sequence 626, and partial cDNA 614c1 (the length of which varies depending on the binding site of target-specific primer 648 in cDNA 614c). PCR1 600c4 may include amplifying the 3' region of single-stranded, extended barcoded nucleic acid molecule 620cd comprising universal oligonucleotide primer 646 and target-specific primer 650, thereby generating a single-labeled nucleic acid molecule 620cas1 comprising first molecular label 622, cell label 624, first universal sequence 626 and partial antisense cDNA 614cas1 (the length of which varies depending on the binding site of target-specific primer 650 in cDNA 614c).PCR1 600c4 may include 1 to 30 cycles (eg, 15 cycles).

[0186] The workflow may include a second round of amplification ("PCR2") 600c5 using universal oligonucleotide primer 646 and nested target-specific primers (e.g., target-specific primer 652 and / or target-specific primer 654). Target-specific primer 652 and / or target-specific primer 654 may include an overhang that may include, or be, second universal sequence 638 (e.g., read 2 sequence, universal PCR handle), for example. PCR2 600c5 may include amplifying single-stranded nucleic acid molecule 620c1 that includes universal oligonucleotide primer 646 and nested target-specific primer 654, thereby generating single-labeled nucleic acid molecule 620c2 that includes first molecular label 622, cell label 624, first universal sequence 626, second universal sequence 638, and partial cDNA 614c2 (the length of which varies depending on the binding site of nested target-specific primer 654 in partial cDNA 614c1). PCR2 600c5 may include amplifying a single-labeled nucleic acid molecule 620cas1 comprising a universal oligonucleotide primer 646 and a nested target-specific primer 652, thereby generating a single-labeled nucleic acid molecule 620cas2 comprising a first molecular label 622, a cell label 624, a first universal sequence 626, a second universal sequence 638, and a partial antisense cDNA 614cas2 (the length of which varies depending on the binding site of the nested target-specific primer 652 in the partial antisense cDNA 614cas1). PCR2 600c5 may include 1 to 30 cycles (e.g., 15 cycles). In some embodiments, the target-specific primers 648, 650, 652, and / or 654 bind to constant, variable, diversity, and / or junction regions of an immune receptor.

[0187] The workflow may include a third round of amplification ("PCR3") 600c6. PCR3 600c6 may include library amplification of single-labeled nucleic acid molecule 620cas2 and / or single-labeled nucleic acid molecule 620c2 with sequencing library amplification primers 656 and 658. Sequencing library amplification primers 656 and 658 may anneal to first universal sequence 626 and second universal sequence 638 (or their complements), respectively. PCR3 600c6 may add sequencing adapters (e.g., P5 640 and P7 642) and sample indexes 644 (e.g., i5, i7) via overhangs in sequencing library amplification primers 656 and 658. Library amplicons 620cas3 and / or 620c3 may be sequenced and subjected to downstream methods of the present disclosure. Sequencing using 150 bp x 2 sequencing can reveal cellular labels, unique molecular labels and / or genes (or partial sequences of genes) on Read 1, genes (or partial sequences of genes) on Read 2, and sample indices on Index 1 read and / or Index 2 read. PCR3 600c6 can include 1 to 30 cycles (e.g., 15 cycles).

[0188] In some embodiments, 3' and / or 5' expression profiling of immune receptor V(D)J regions may be performed. In some embodiments, both phenotypic markers and immune receptor V(D)J sequences of T cells and / or B cells in a single-cell platform may be investigated. In some embodiments, both 3' and 5' information of those transcripts may be captured in a single experiment. The methods disclosed herein may enable V(D)J detection (e.g., hypermutation) of both T cells and B cells. In some embodiments, both the 3' and 5' regions of the extended barcoded nucleic acid molecule 620cd are amplified. In some embodiments, only the 5' region of the extended barcoded nucleic acid molecule 620cd is amplified. In some embodiments, only the 3' region of the extended barcoded nucleic acid molecule 620cd is amplified. In some embodiments, one or more of the amplification reactions include multiplex PCR. For example, both the 3' and 5' regions of the extended barcoded nucleic acid molecule 620cd may be amplified simultaneously (e.g., multiplex PCR). In some embodiments, the workflow comprises multiplex PCR using a panel of target-specific PCR1 primers and / or a panel of target-specific PCR2 primers. In some embodiments, the targets comprise BCR, TCR, and / or immune-related transcripts.

[0189] Intermolecular hybridization of barcoded nucleic acid molecules with barcoded nucleic acid molecules In some embodiments, hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules comprises intermolecular hybridization of the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of a different barcoded nucleic acid molecule among the plurality of barcoded nucleic acid molecules. In some embodiments, the sequence of the second molecular label differs from the sequence of the first molecular label, and the second molecular label is not a complement of the first molecular label.

[0190] The workflow may involve intermolecular hybridization of a single-stranded barcoded nucleic acid molecule 621 with a separate barcoded nucleic acid molecule 628, as shown in the non-limiting exemplary schematic diagrams of Figures 6E-6F. The separate barcoded nucleic acid molecule 628 may include cDNA 630c, a second molecular label 632, a cell label 624, and a first universal sequence 626. The sequence of the second molecular label 632 of the barcoded nucleic acid molecule 628 may be different from the sequence of the first molecular label 622 of the single-stranded barcoded nucleic acid molecule 621 (e.g., not a complement). The target binding region 604, cell label 624, and / or first universal sequence 626 of the barcoded nucleic acid molecule 628 may be the same as (or a complement of) the target binding region 604, cell label 624, and / or first universal sequence 626 of the single-stranded barcoded nucleic acid molecule 621. In some embodiments, the workflow may include intermolecular hybridization 600d1 of the complement of the target binding region 608 of the single-stranded barcoded nucleic acid molecule 621 with the target binding region 604 of the barcoded nucleic acid molecule 628. The workflow may include extending (600d2) the 3' end of the single-stranded barcoded nucleic acid molecule 621 to generate an extended barcoded nucleic acid molecule 620d. The extended barcoded nucleic acid molecule 620d may include a complement (e.g., reverse complement) 632rc of a second molecular label, a complement (e.g., reverse complement) 624rc of a cell label, and / or a complement (e.g., reverse complement) 626rc of a first universal sequence. The workflow may include denaturing (600d3) the extended barcoded nucleic acid molecule 620d to generate a single-stranded extended barcoded nucleic acid molecule 620dd. In some embodiments, intermolecular hybridization 600d1 and / or extension 600d2 are performed in the presence of a high salt buffer and / or PEG. In some embodiments, extension is performed using a DNA polymerase that lacks at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity (e.g., Klenow fragment).

[0191] The single-stranded, extended barcoded nucleic acid molecule 620dd includes barcodes (e.g., cell and molecular labels) at both the 5' and 3' ends of the target nucleic acid molecule (e.g., transcript), thereby enabling more extensive analysis of the target nucleic acid molecule for sequence identification, transcript enumeration, alternative splicing analysis, mutation screening, and / or full-length sequencing compared to analysis of a target nucleic acid molecule having only one barcode at one end. The single-stranded, extended barcoded nucleic acid molecule 620dd can serve as a template for one or more amplification reactions (e.g., PCR), such as, for example, the non-limiting exemplary amplification schemes shown in Figures 6G-6H. Amplification can include target-specific (e.g., gene-specific) cDNA amplification. For example, single-stranded, extended barcoded nucleic acid molecule 620dd may undergo a first round of amplification ("PCR1") 600d4 using a universal oligonucleotide primer 646 comprising the sequence of a first universal sequence (or its complement) and a universal oligonucleotide primer 646 comprising a target-specific primer (e.g., target-specific primer 648 and / or target-specific primer 650). PCR1 600d4 may include amplifying a 5' region of single-stranded, extended barcoded nucleic acid molecule 620dd comprising universal oligonucleotide primer 646 and target-specific primer 648, thereby generating single-labeled nucleic acid molecule 620d1 comprising first molecular label 622, cell label 624, first universal sequence 626, and partial cDNA 614c1 (the length of which varies depending on the binding site of target-specific primer 648 in cDNA 614c). PCR1 600d4 may include amplifying the 3' region of single-stranded, extended barcoded nucleic acid molecule 620dd, which comprises universal oligonucleotide primer 646 and target-specific primer 650, thereby generating single-labeled nucleic acid molecule 620das1, which comprises second molecular label 632, cell label 624, first universal sequence 626, and partial antisense cDNA 614cas1 (the length of which varies depending on the binding site of target-specific primer 650 in cDNA 614c).PCR1 600d4 may include 1 to 30 cycles (eg, 15 cycles).

[0192] The workflow may include a second round of amplification ("PCR2") 600d5 using universal oligonucleotide primer 646 and nested target-specific primers (e.g., target-specific primer 652 and / or target-specific primer 654). Target-specific primer 652 and / or target-specific primer 654 may include an overhang that may include, or be, second universal sequence 638 (e.g., read 2 sequence, universal PCR handle), for example. PCR2 600d5 may include amplifying single-labeled nucleic acid molecule 620d1 that includes universal oligonucleotide primer 646 and nested target-specific primer 654, thereby generating single-labeled nucleic acid molecule 620d2 that includes first molecular label 622, cell label 624, first universal sequence 626, second universal sequence 638, and partial cDNA 614c2 (the length of which varies depending on the binding site of nested target-specific primer 654 in cDNA 614c1). PCR2 600d5 may include amplifying a single-labeled nucleic acid molecule 620das1, which includes a universal oligonucleotide primer 646 and a nested target-specific primer 652, thereby generating a single-labeled nucleic acid molecule 620das2, which includes a second molecular label 632, a cell label 624, a first universal sequence 626, a second universal sequence 638, and a partial antisense cDNA 614cas2 (the length of which varies depending on the binding site of the nested target-specific primer 652 in the partial antisense cDNA 614cas1). PCR2 600d5 may include 1 to 30 cycles (e.g., 15 cycles). In some embodiments, the target-specific primers 648, 650, 652, and / or 654 bind to the constant region, variable region, diversity region, and / or junction region of an immune receptor.

[0193] The workflow may include a third round of amplification ("PCR3") 600d6. PCR3 600d6 may include library amplification of single-labeled nucleic acid molecule 620das2 and / or single-labeled nucleic acid molecule 620d2 with sequencing library amplification primers 656 and 658. Sequencing library amplification primers 656 and 658 may anneal to first universal sequence 626 and second universal sequence 638 (or their complements), respectively. PCR3 600d6 may add sequencing adapters (e.g., P5 640 and P7 642) and sample indexes 644 (e.g., i5, i7) via overhangs in sequencing library amplification primers 656 and 658. Library amplicons 620das3 and / or 620d3 may be sequenced and subjected to downstream methods of the present disclosure. Sequencing using 150 bp x 2 sequencing can reveal cell labels, unique molecular labels and / or genes (or partial sequences of genes) on Read 1, genes (or partial sequences of genes) on Read 2, and sample indices on Index 1 read and / or Index 2 read. PCR3 600d6 can include 1 to 30 cycles (e.g., 15 cycles).

[0194] In some embodiments, 3' and / or 5' expression profiling of immune receptor V(D)J regions may be performed. In some embodiments, both phenotypic markers and immune receptor V(D)J sequences of T cells and / or B cells in a single-cell platform may be investigated. In some embodiments, both 3' and 5' information of those transcripts may be captured in a single experiment. The methods disclosed herein may enable V(D)J detection of both T cells and B cells (e.g., hypermutation). In some embodiments, both the 3' and 5' regions of the extended barcoded nucleic acid molecule 620dd are amplified. In some embodiments, only the 5' region of the extended barcoded nucleic acid molecule 620dd is amplified. In some embodiments, only the 3' region of the extended barcoded nucleic acid molecule 620dd is amplified. In some embodiments, one or more of the amplification reactions comprise multiplex PCR. For example, both the 3' and 5' regions of the extended barcoded nucleic acid molecule 620dd may be amplified simultaneously (e.g., multiplex PCR). In some embodiments, the workflow comprises multiplex PCR using a panel of target-specific PCR1 primers and / or a panel of target-specific PCR2 primers. In some embodiments, the targets comprise BCR, TCR, and / or immune-related transcripts.

[0195] Intermolecular hybridization of barcoded nucleic acid molecules with oligonucleotide barcodes In some embodiments, hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of an oligonucleotide barcode among the plurality of oligonucleotide barcodes comprises intermolecular hybridization of the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of an oligonucleotide barcode among the plurality of oligonucleotide barcodes. In some embodiments, the second molecular label is different from the first molecular label, and the second molecular label is not a complement of the first molecular label. In some embodiments, the method comprises extending the 3' ends of the oligonucleotide barcodes hybridized to the complement of the target binding region of the barcoded nucleic acid molecule to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a complement of the first molecular label and a second molecular label. In some embodiments, the sequence of the second molecular label is different from the sequence of the first molecular label, and the second molecular label is not a complement of the first molecular label.

[0196] The workflow may involve intermolecular hybridization of a single-stranded barcoded nucleic acid molecule 621 with a distinct oligonucleotide barcode 634, as shown in the non-limiting exemplary schematic diagrams of Figures 6I-6J. The distinct oligonucleotide barcode 634 may include a second molecular label 636, a cell label 624, and a first universal sequence 626. The sequence of the second molecular label 636 of the oligonucleotide barcode 634 may be different from the sequence of the first molecular label 622 of the single-stranded barcoded nucleic acid molecule 621 (e.g., not a complement). The target binding region 604, cell label 624, and / or first universal sequence 626 of the oligonucleotide barcode 634 may be the same as (or a complement of) the target binding region 604, cell label 624, and / or first universal sequence 626 of the single-stranded barcoded nucleic acid molecule 621. The workflow, in some embodiments, may include intermolecular hybridization 600e1 of the complement of the target binding region 608 of the single-stranded barcoded nucleic acid molecule 621 with the target binding region 604 of the oligonucleotide barcode 634. The workflow may include extending (600e2) the 3' end of the single-stranded barcoded nucleic acid molecule 621 to generate an extended barcoded nucleic acid molecule 620e1. The extended barcoded nucleic acid molecule 620e1 may include a complement (e.g., reverse complement) 636rc of the second molecular label, a complement (e.g., reverse complement) 624rc of the cell label, a complement (e.g., reverse complement) 626rc of the first universal sequence, and / or cDNA 614c. The workflow may include denaturing (600e3) extended barcoded nucleic acid molecule 620e1 to generate single-stranded extended barcoded nucleic acid molecule 620e1d. The workflow may include extending (600e2) the 3' end of oligonucleotide barcode 634 to generate extended barcoded nucleic acid molecule 620e2.The extended barcoded nucleic acid molecule 620e2 may include a complement (e.g., reverse complement) 622rc of the first molecular label, a complement (e.g., reverse complement) 624rc of the cell label, a complement (e.g., reverse complement) 626rc of the first universal sequence, and / or an antisense cDNA 614cas. The workflow may include denaturing (600e3) the extended barcoded nucleic acid molecule 620e2 to generate a single-stranded extended barcoded nucleic acid molecule 620e2d. In some embodiments, intermolecular hybridization 600e1 and / or extension 600e2 are performed in the presence of a high salt buffer and / or PEG. In some embodiments, extension is performed using a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity (e.g., Klenow fragment).

[0197] Single-stranded extended barcoded nucleic acid molecule 620e1d and single-stranded extended barcoded nucleic acid molecule 620e2d contain barcodes (e.g., cell and molecular labels) at both the 5' and 3' ends of the target nucleic acid molecule (e.g., transcript), thereby enabling more extensive analysis of the target nucleic acid molecule for sequence identification, transcript counting, alternative splicing analysis, mutation screening, and / or full-length sequencing compared to analysis of a target nucleic acid molecule having only one barcode at one end. Single-stranded extended barcoded nucleic acid molecule 620e1d and single-stranded extended barcoded nucleic acid molecule 620e2d can serve as templates for one or more amplification reactions (e.g., PCR). Amplification can include target-specific (e.g., gene-specific) cDNA amplification. In some embodiments, single-stranded extended barcoded nucleic acid molecule 620e1d and / or single-stranded extended barcoded nucleic acid molecule 620e2d may undergo two or more PCR amplifications (e.g., PCR1 600d4, PCR2 600d5, and / or PCR3 600d6 as shown in Figures 6G-6H). In some embodiments, single-stranded extended barcoded nucleic acid molecule 620e1d and / or single-stranded extended barcoded nucleic acid molecule 620e2d may serve as a template for a single amplification, such as, for example, the non-limiting exemplary amplification scheme (PCR 600e4) shown in Figure 6K. PCR 600e4 may add sequencing adapters (e.g., P5 640 and P7 642) and sample index 644 (e.g., i5, i7) via overhangs in primers 660, 662, and 664.PCR 600e4 may include amplifying single-stranded, extended barcoded nucleic acid molecule 620e1d, which includes primer 664 (annealing to the first universal sequence or its complement) and target-specific primer 660, thereby generating single-labeled nucleic acid molecule 620e1c, which includes first molecular label 622, cell label 624, first universal sequence 626, and partial cDNA 614c1e (the length of which varies depending on the binding site of target-specific primer 660 in cDNA 614c). PCR 600e4 may include amplifying single-stranded, extended barcoded nucleic acid molecule 620e2d, which includes primer 664 (annealing to the first universal sequence or its complement) and target-specific primer 662, thereby generating single-labeled nucleic acid molecule 620e2c, which includes second molecular label 636, cell label 624, first universal sequence 626, and partial antisense cDNA 614cas1e (whose length varies depending on the binding site of target-specific primer 662 in antisense cDNA 614cas1e). Library amplicons 620e1c and / or 620e2c can be sequenced and subjected to downstream methods of the present disclosure. Sequencing using 150 bp × 2 sequencing may reveal cell labels, unique molecular labels, and / or genes (or partial sequences of genes) on Read 1, genes (or partial sequences of genes) on Read 2, and sample indices on Index 1 read and / or Index 2 read. PCR 600e4 can include 1 to 30 cycles (e.g., 15 cycles). In some embodiments, target-specific primers 660 and / or 662 bind to constant regions, variable regions, diversity regions, and / or junction regions of immune receptors.

[0198] In some embodiments, 3' and / or 5' expression profiling of immune receptor V(D)J regions may be performed. In some embodiments, both phenotypic markers and immune receptor V(D)J sequences of T cells and / or B cells in a single-cell platform may be investigated. In some embodiments, both 3' and 5' information of transcripts may be captured in a single experiment. The methods disclosed herein may enable V(D)J detection (e.g., hypermutation) in both T cells and B cells. In some embodiments, both the 3' and 5' regions of extended barcoded nucleic acid molecules 620e1d and / or 620e2d are amplified. In some embodiments, only the 5' region of extended barcoded nucleic acid molecules 620e1d and / or 620e2d is amplified. In some embodiments, only the 3' region of extended barcoded nucleic acid molecules 620e1d and / or 620e2d is amplified. In some embodiments, one or more of the amplification reactions comprise multiplex PCR. For example, both the 3' and 5' regions of extended barcoded nucleic acid molecules 620e1d and / or 620e2d can be amplified simultaneously (e.g., multiplex PCR). In some embodiments, the workflow includes multiplex PCR using a panel of target-specific PCR1 primers and / or a panel of target-specific PCR2 primers. In some embodiments, the targets include BCR, TCR, and / or immune-related transcripts.

[0199] Immune repertoire profiling In some embodiments, a method for 3' and / or 5' expression profiling of the V(D)J region of an immune receptor is provided. In some embodiments, the sample comprises a single cell. In some embodiments, the sample comprises a plurality of cells, a plurality of single cells, a tissue, a tumor sample, or any combination thereof. The single cell may comprise an immune cell. In some embodiments, the immune cell is a B cell or a T cell. In some embodiments, the single cell may comprise a circulating tumor cell. In some embodiments, each oligonucleotide barcode may comprise a first universal sequence. In some embodiments, the plurality of extended barcoded nucleic acid molecules comprises the first universal sequence and a complement of the first universal sequence. In some embodiments, amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of copies of the extended barcoded nucleic acid molecule comprises using a primer capable of hybridizing to the first universal sequence, or its complement. In some embodiments, amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules includes using a primer capable of hybridizing to a first universal sequence or its complement and an amplification primer. In some embodiments, the amplification primer is a target-specific primer. In some such embodiments, the target-specific primer specifically hybridizes to an immune receptor. For example, the target-specific primer may specifically hybridize to a constant region of the immune receptor, a variable region of the immune receptor, a diversity region of the immune receptor, a junction between the variable region and the diversity region of the immune receptor, or any combination thereof. The immune receptor may be a T cell receptor (TCR) and / or a B cell receptor (BCR). The TCR may include a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof. The BCR may include a BCR heavy chain and / or a BCR light chain.

[0200] The method may include obtaining sequence information of a plurality of extended barcoded nucleic acid molecules, or products thereof. Obtaining sequence information may include attaching sequencing adapters to the plurality of extended barcoded nucleic acid molecules, or products thereof. Obtaining sequence information may include attaching sequencing adapters to a plurality of single-labeled nucleic acid molecules, or products thereof.

[0201] The step of obtaining sequence information may include obtaining sequence information of the BCR light chain and BCR heavy chain of the single cell. The sequence information of the BCR light chain and BCR heavy chain may include the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the BCR light chain and / or BCR heavy chain. The method may include pairing the BCR light chain and BCR heavy chain of the single cell based on the obtained sequence information. The sample may include a plurality of single cells, and the method may include pairing the BCR light chain and BCR heavy chain of at least 50% of the single cells based on the obtained sequence information. In some embodiments, the percentage of single cells of a sample in which a BCR light chain and a BCR heavy chain are paired according to the methods provided herein is 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, %,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, It may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values, or about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56 %, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of single cells of a sample in which the BCR light chain and BCR heavy chain are paired according to the methods provided herein is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.0001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, It can be 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0202] The step of obtaining sequence information may include obtaining sequence information of the TCR alpha chain and TCR beta chain of the single cell. In some embodiments, the sequence information of the TCR alpha chain and TCR beta chain may include the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the TCR alpha chain and / or TCR beta chain. In some embodiments, the method may include pairing the TCR alpha chain and TCR beta chain of the single cell based on the obtained sequence information. In some embodiments, the sample may include a plurality of single cells, and the method may include pairing the TCR alpha chain and TCR beta chain of at least 50% of the single cells based on the obtained sequence information. In some embodiments, the percentage of single cells of a sample in which the TCR alpha chain and TCR beta chain are paired according to the methods provided herein is 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 10 %, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values, or may be about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56 %, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of single cells of a sample in which the TCR alpha chain and TCR beta chain are paired according to the methods provided herein is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, %, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0203] The step of obtaining sequence information may include obtaining sequence information of the TCR gamma chain and the TCR delta chain of the single cell. The sequence information of the TCR gamma chain and the TCR delta chain may include the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the TCR gamma chain and / or the TCR delta chain. The method may include pairing the TCR gamma chain and the TCR delta chain of the single cell based on the obtained sequence information. The sample may include a plurality of single cells, and the method may include pairing the TCR gamma chain and the TCR delta chain of at least 50% of the single cells based on the obtained sequence information. In some embodiments, the percentage of single cells of a sample in which the TCR delta chain and TCR gamma chain are paired according to the methods provided herein is 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 10 %, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values, or may be about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56 %, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of single cells of a sample in which the TCR delta chain and the TCR gamma chain are paired according to the methods provided herein is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101 %, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0204] Kit for barcoding nucleic acid targets at the 5' and 3' ends The present disclosure includes kits. In some embodiments, the kits include a plurality of oligonucleotide barcodes, each of the plurality of oligonucleotide barcodes comprising a molecular label and a target binding region, wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences; a reverse transcriptase; a template switching oligonucleotide comprising a target binding region, or a portion thereof; and a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. In some embodiments, the DNA polymerase comprises a Klenow fragment. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is murine leukemia virus (MLV) reverse transcriptase. In some embodiments, the viral reverse transcriptase is Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the template switch oligonucleotide comprises one or more 3' ribonucleotides, e.g., three 3' ribonucleotides. In some embodiments, the 3' ribonucleotide comprises a guanine. In some embodiments, the kit comprises one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof.

[0205] In some embodiments, the kit includes a buffer. In some embodiments, the kit includes a cartridge. In some embodiments, the kit includes one or more reagents for a reverse transcription reaction. In some embodiments, the kit includes one or more reagents for an amplification reaction. In some embodiments, the target binding regions include gene-specific sequences, oligo(dT) sequences, random multimers, or any combination thereof. In some embodiments, the oligonucleotide barcodes include identical sample labels and / or identical cell labels. In some embodiments, each sample label and / or cell label of the plurality of oligonucleotide barcodes includes at least six nucleotides. In some embodiments, each molecular label of the plurality of oligonucleotide barcodes includes at least six nucleotides. In some embodiments, at least one of the plurality of oligonucleotide barcodes is immobilized on a synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially immobilized on a synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is encapsulated within a synthetic particle. In some embodiments, at least one of the plurality of oligonucleotide barcodes is partially encapsulated within a synthetic particle. In some embodiments, the synthetic particle is disintegratable. In some embodiments, the synthetic particles comprise beads, such as 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. In some embodiments, the synthetic 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.In some embodiments, the synthetic particle comprises a collapsible hydrogel particle. In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and / or the support functional group and the linker functional group are associated with each other. In some embodiments, the linker functional group and the support functional group are independently selected from the group consisting of C6, biotin, streptavidin, primary amines, aldehydes, ketones, and any combination thereof. [Example]

[0206] Certain aspects of the above embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. The non-limiting exemplary V(D)J protocol described below was used to demonstrate the generation of sequencing libraries for both the 3' and 5' ends of the mRNA targets of the targeting panel.

[0207] BD Rhapsody® Cell Capture and Reverse Transcription 1. Prepare a single cell suspension of the sample cells. 2. Follow standard BD Rhapsody® protocol for harvesting and capturing single cells by washing beads and placing beads on ice. 3. Make the template switch reaction mix according to Table 1 below. [Table 1] 4. Place beads on magnet, remove supernatant and resuspend beads in 200 uL of reaction mix. 5. Place the tube on a thermomixer for 30 minutes at 25°C, followed by 1.5 hours at 42°C at 1200 rpm. Place on ice after the reaction is complete. 6. Place the beads on a magnet and remove the supernatant. 7. Resuspend the beads in 1 mL of TE buffer. 8. Heat the beads to 95°C for 2 minutes to denature the mRNA. 9. Place the beads on a magnet and remove the supernatant. 10. Resuspend the beads in 1 mL of TE buffer. 11. Heat the beads to 95°C for 2 minutes to denature the mRNA. 12. Place the beads on a magnet and remove the supernatant. 13. Resuspend the beads in 2 mL of pre-warmed (37° C.) HT1 buffer (Illumina, San Diego, CA).

[0208] Self-hybridization 1. Shake the tube at 1200 rpm for 5 minutes at 37°C, followed by 25 minutes at 25°C. Then place on ice. 2. Wash the beads once with 1 mL of HT1 buffer.

[0209] Klenow extension 1. Prepare the Klenow extension reaction mix shown in Table 2 below. [Table 2] 2. Place the beads on a magnet and remove the supernatant. 3. Resuspend the beads in 200 uL of Klenow extension reaction mix. 4. Place in thermomixer at 37°C for 30 minutes at 1200 rpm. 5. Wash once with 1 mL of TE.

[0210] ExoI processing 1. Prepare the ExoI reaction mix according to Table 3 below. [Table 3] 2. Place the beads on a magnet and remove the supernatant. 3. Resuspend the beads in 200 uL of ExoI reaction mix. 4. Place the tube in a thermomixer at 1200 rpm and 37C for 30 minutes. 5. Transfer the tube to a thermomixer at 80°C for 20 minutes without shaking. 6. Place the tube on ice for approximately 1 minute. 7. Place the beads on the magnet. 8. Remove the supernatant and resuspend the beads in 200 uL of Bead Resuspension Buffer.

[0211] PCR1 amplification 1. Prepare PCR1 master mix according to Table 4 below: [Table 4] 2. (Optional) Subsample beads. 3. Place the tube containing the beads on a magnet and remove the supernatant. 4. Resuspend the beads in 200uL of PCR1 reaction mix. Gently pipette up and down to mix thoroughly. 5. Divide (4) evenly into 0.2 ml PCR tubes (i.e., approximately 50 ul ± 5 ul per tube). 6. In the post-PCR room, run the following PCR protocol: 95°C for 3 minutes, 15 cycles of (95°C for 30 seconds, 60°C for 3 minutes, 72°C for 1 minute), 72°C for 5 minutes. Hold at 4°C. 7. After PCR, combine the PCR1 product and beads in a LoBind 1.5 ml microcentrifuge tube. 8. Place the tube on a 1.5 ml magnet and pipet the PCR1 product into a new tube.

[0212] PCR1 cleanup 1. Add 200ul of Ampure XP beads (1x the volume of the PCR product) to the PCR1 product. Mix well. 2. Incubate at room temperature for 5 minutes. 3. Prepare fresh 80% ethanol (e.g., 800 ul ethanol and 200 ul DNase / RNase free water). 4. Place the tube containing the Ampure beads on a 1.5 ml tube magnet for approximately 1-2 minutes. After all the beads have collected on the side of the tube, remove the supernatant. 5. After all the beads have collected on the side of the tube, remove the supernatant. 6. While the tube is on the magnet, add 500ul of 80% ethanol to wash the bead pellet. 7. Remove as much ethanol as possible. 8. Repeat the 80% ethanol wash once for a total of two washes. 9. Allow the Ampure beads to air dry on the magnet with the lid open until no visible droplets are present (approximately 3-5 minutes). 10. While the tube is on the magnet, add 500ul of 80% ethanol to wash the bead pellet. 11. Remove as much ethanol as possible. 12. Repeat the 80% ethanol wash once for a total of two washes. 13. Allow the Ampure beads to air dry on the magnet with the lid open until no visible droplets are present (approximately 3-5 minutes). 14. Resuspend Ampure beads in 30 ul of elution buffer. 15. Place the 1.5 ml tube on the magnet. 16. Transfer the supernatant to a new 1.5 ml tube. This is the purified PCR1 product. Store at 4 C or on ice if the next step is to be performed the same day, or store at -20°C until use.

[0213] PCR2 amplification 1. In the Pre-PCR area, prepare the following reaction mix: [Table 5]

[0214] [Table 6]

[0215] [Table 7]

[0216] [Table 8] 2. Transfer the reaction mix to the post-PCR area. 3. Add 5 ul of cleaned up PCR1 product to 45 ul of reaction mix. 4. Run the following PCR protocol in the thermal cycler in the post-PCR area: 95°C for 3 minutes, 15 cycles of (95°C for 30 seconds, 60°C for 3 minutes, 72°C for 1 minute), 72°C for 5 minutes.

[0217] PCR2 cleanup 1. For TCR and BCR products, add 30ul of Ampure XP beads (0.6x the volume of the PCR product) to the PCR1 product. For IR3' and 5', add 50ul of Ampure XP beads (1x the volume). Mix well. 2. Incubate at room temperature for 5 minutes. 3. Prepare fresh 80% ethanol (e.g., 800 ul ethanol and 200 ul DNase / RNase free water). 4. Place the tube containing the Ampure beads on a 1.5 ml tube magnet for approximately 1-2 minutes. After all the beads have collected on the side of the tube, remove the supernatant. 5. While the tube is on the magnet, add 200 ul of 80% ethanol to wash the bead pellet. 6. Remove as much ethanol as possible. 7. Repeat the 80% ethanol wash once for a total of two washes. 8. Allow the Ampure beads to air dry on the magnet with the lid open until no droplets are evident. 9. Resuspend the beads in 30 ul of elution buffer. 10. Place the 1.5 ml tube on the magnet. 11. Transfer the supernatant to a new 1.5 ml tube. This is the purified PCR2 product. Store at 4°C or on ice if the next step is to be performed the same day, or store at -20°C until use. 12. Use the Qubit DNA HS Assay to measure the amount of eluted DNA and assess whether product dilution is necessary for the next PCR. The PCR2 product must be diluted to 10 ng / ul or less using elution buffer before proceeding to the final PCR to avoid over-amplification.

[0218] Indexed PCR 1. In the Pre-PCR area, prepare the following reaction mix as shown in Table 9. [Table 9] 2. Transfer the reaction mix to the post-PCR area. 3. Add 3 ul of cleaned up PCR2 product to 47 ul of reaction mix. 4. Run the following PCR protocol in the Post PCR area: 95°C for 5 minutes, 8 cycles of (98°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds), 72°C for 1 minute.

[0219] Final PCR cleanup 1. Add 30ul of Ampure XP beads (0.6x the volume of the PCR product) to the PCR product. Mix well. 2. Incubate at room temperature for 5 minutes. 3. Prepare fresh 80% ethanol (e.g., 800 ul ethanol and 200 ul DNase / RNase free water). 4. Place the tube containing the Ampure beads on a 1.5 ml tube magnet for approximately 1-2 minutes. After all the beads have collected on the side of the tube, remove the supernatant. 5. While the tube is on the magnet, add 200 ul of 80% ethanol to wash the bead pellet. 6. Remove as much ethanol as possible. 7. Repeat the 80% ethanol wash once for a total of two washes. 8. Allow the Ampure beads to air dry on the magnet with the lid open until no droplets are evident. 9. Resuspend the beads in 30 ul of elution buffer. 10. Place the 1.5 ml tube on the magnet. 11. Transfer the supernatant to a new 1.5 ml tube. This is the purified PCR2 product. Store at 4°C or on ice if the next step is to be performed the same day, or store at -20°C until use. 12. Use the Qubit DNA HS Assay to measure the amount of eluted DNA and assess whether product dilution is necessary for the next PCR. The PCR2 product must be diluted to 10 ng / ul or less using elution buffer before proceeding to the final PCR to avoid over-amplification.

[0220] Example 1 V(D)J Protocol This example demonstrates generating sequencing libraries for both the 3' and 5' ends of mRNA targets in a targeted panel. In this example, we generated sequencing libraries for both the 3' and 5' ends of mRNA targets from a targeting panel that also included the V(D)J regions of T cell receptors and immunoglobulin genes. In addition to simultaneous analysis of 5' and 3' Universal Molecular Index (UMI) counts, we identified CDR3 rearrangement patterns in lymphocytes from peripheral blood mononuclear cells of healthy donors.

[0221] Figures 8A-8C show non-limiting, exemplary experimental results of capturing and sequencing the 5' T cell receptor (TCR) V(D)J region using the V(D)J protocol. The V(D)J hairpin protocol can involve 3' and 5' amplifications performed on the same beads for captured mRNA molecules from single, resting peripheral blood mononuclear cells (PBMCs). Figure 8A shows cell type annotation. Figures 8B and 8C show the expression profiles of TCR alpha and TCR beta, respectively, using 5' amplifications performed with this V(D)J protocol. The TCR alpha / beta chain pairing efficiency was 37.9%, which is comparable to other platforms, such as Clonetech's scTCR profiling kit. Table 10 shows the TCR alpha / beta pairing achieved using this V(D)J protocol.

[0222] [Table 10]

[0223] Figures 9A-9B show non-limiting exemplary plots demonstrating improved 5' V(D)J detection sensitivity using the improved V(D)J protocol. Ethylene glycol was added to aid in the reduction of secondary structure during reverse transcription (RT). Hybridization time, buffer, and template switching (TS) oligo-dT length were modified to improve sensitivity. Four libraries were generated and sequenced together: 5' TCR, 5' BCR, 5' 30-plex immune panel, and 3' immune response panel. A more stringent Ampure cleanup (0.6X) was performed. Figure 9B shows a bioanalyzer plot achieved using the improved V(D)J protocol, while Figure 9A shows a bioanalyzer plot achieved for the V(D)J protocol described with reference to Figure 8.

[0224] Figures 10A-10E show non-limiting, exemplary experimental results of improving 5' V(D)J detection sensitivity using the improved V(D)J protocol described with respect to Figure 9. Figure 10A shows cell type annotation. Figures 10B-10E show expression profiles obtained for TCR alpha, TCR beta, IGKC, and IGLC using the improved V(D)J protocol. Compared to the protocol described with respect to Figure 8, TCR alpha / beta pairing efficiency was improved from 37.9% to 52%. 5' heavy and light chain mRNA molecules in B cells were successfully detected. Tables 11 and 12 show TCR alpha / beta pairing and BCR heavy chain / light chain pairing, respectively, using the improved V(D)J protocol.

[0225] [Table 11] [Table 12]

[0226] Figures 11A-F are non-limiting exemplary plots showing detection of 5' B-cell heavy chains using the improved V(D)J protocol, which has improved sensitivity compared to the expression profiles of IGHM, IGHD, and IGHA determined using 3' amplification (Figures 11A, 11C, 11E) and 5' amplification (Figures 11B, 11D, 11F). Figures 12A-12F are non-limiting exemplary plots comparing the expression profiles of CD3D, CD8A, and HLA-DR determined using 3' amplification (Figures 12A, 12C, 12E) and 5' amplification (Figures 12B, 12D, 12F). Taken together, the data indicate that the ability to profile both 3' and 5' mRNA transcript information can expand the flexibility and potential of 3' single-cell RNA sequencing platforms. While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

[0227] Those skilled in the art will recognize that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be realized in differing order. Furthermore, the outlined steps and operations are provided only as examples, and some of the steps and operations may be arbitrarily combined into fewer steps and operations or expanded into additional steps and operations without detracting from the essential elements of the embodiments of the present disclosure. In connection with the use of virtually any plural and / or singular term herein, those skilled in the art can convert from plural to singular and / or from singular to plural where appropriate in the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0228] In general, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Furthermore, where a specific number of introduced claim recitations is intended, such intention will be explicitly set forth in the claim; it will be understood by those skilled in the art that, in the absence of such a recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means that any particular claim containing such introduced claim recitation is limited to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), nor should the use of definite articles used to introduce claim recitations.Furthermore, even if a particular number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., an unqualified recitation such as "two recitations" without other modifiers means at least two recitations, or more than two recitations). Furthermore, when a convention similar to "such as at least one of A, B, and C" is used, it is generally intended that such a configuration be understood by one of skill in the art (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). When a convention similar to "such as at least one of A, B, or C" is used, it is generally intended that such construction have the meaning that one of ordinary skill in the art would understand that convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Furthermore, it will be understood by those skilled in the art that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B." Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0229] As will be understood by those skilled in the art, for all purposes, e.g., with respect to the provision of a specification, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any recited range is readily recognizable as fully descriptive and that range can be divided into at least 2, 3, 4, 5, 10, etc. divisions. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. Similarly, as will be understood by those skilled in the art, all expressions such as "up to," "at least," etc. refer to ranges that are inclusive of the recited number and that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so forth.

[0230] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for labeling nucleic acid targets in a sample, comprising: contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending a plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target in the presence of a reverse transcriptase and a template switch oligonucleotide comprising a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each comprising a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; The complement of the target binding region of each barcoded nucleic acid molecule is (i) an oligonucleotide barcode among a plurality of oligonucleotide barcodes; (ii) the barcoded nucleic acid molecule itself, and / or (iii) different barcoded nucleic acid molecules among the plurality of barcoded nucleic acid molecules. hybridizing the target binding region of extending the 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of the extended barcoded nucleic acid molecules comprising a first molecular label and a second molecular label; A method comprising:

2. 10. The method of claim 1, comprising determining the copy number of a nucleic acid target in a sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with a plurality of extended barcoded nucleic acid molecules, or products thereof.

3. 1. A method for determining the number of nucleic acid targets in a sample, comprising: contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending a plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target in the presence of a reverse transcriptase and a template switch oligonucleotide comprising a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each comprising a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; The complement of the target binding region of each barcoded nucleic acid molecule is (i) an oligonucleotide barcode among a plurality of oligonucleotide barcodes; (ii) the barcoded nucleic acid molecule itself, and / or (iii) different barcoded nucleic acid molecules among the plurality of barcoded nucleic acid molecules. hybridizing the target binding region of extending the 3' ends of a plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a first molecular label and a second molecular label; determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels having distinct sequences, second molecular labels having distinct sequences, or a combination thereof, associated with the plurality of extended barcoded nucleic acid molecules, or products thereof; A method comprising:

4. amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules, each of which comprises the first molecular label or the second molecular label; 4. The method of claim 2, wherein determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels having distinct sequences associated with a plurality of single-labeled nucleic acid molecules.

5. 5. The method of claim 4, wherein determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on the number of first molecular labels having distinct sequences associated with a plurality of single-labeled nucleic acid molecules.

6. amplifying the plurality of extended bar-coded nucleic acid molecules to generate a plurality of copies of the extended bar-coded nucleic acid molecules; 4. The method of any one of claims 2-3, wherein determining the copy number of the nucleic acid target in the sample comprises determining the copy number of the nucleic acid target in the sample based on (i) the number of first molecular labels with distinct sequences associated with the copies of the plurality of extended barcoded nucleic acid molecules, or products thereof, and / or (ii) the number of second molecular labels with distinct sequences associated with the copies of the plurality of extended barcoded nucleic acid molecules, or products thereof.

7. 1. A method for determining the number of nucleic acid targets in a sample, comprising: contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, each oligonucleotide barcode comprising a molecular label and a target binding region capable of hybridizing to the nucleic acid target; extending a plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target in the presence of a reverse transcriptase and a template switch oligonucleotide comprising a target binding region, or a portion thereof, to generate a plurality of barcoded nucleic acid molecules, each comprising a sequence complementary to at least a portion of the nucleic acid target, a first molecular label, a target binding region, and a complement of the target binding region; The complement of the target binding region of each barcoded nucleic acid molecule is (i) an oligonucleotide barcode among a plurality of oligonucleotide barcodes; (ii) the barcoded nucleic acid molecule itself, and / or (iii) different barcoded nucleic acid molecules among the plurality of barcoded nucleic acid molecules. hybridizing the target binding region of extending the 3' ends of a plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a first molecular label and a second molecular label; amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules, each of which comprises a first molecular label or a second molecular label; determining the copy number of the nucleic acid target in the sample based on the number of second molecular labels having distinct sequences associated with the plurality of single-labeled nucleic acid molecules; A method comprising:

8. 8. The method of claim 7, comprising determining the copy number of a nucleic acid target in a sample based on the number of first molecular labels having distinct sequences associated with a plurality of single-labeled nucleic acid molecules.

9. 9. The method of any one of claims 1 to 8, comprising denaturing the plurality of barcoded nucleic acid molecules prior to hybridizing the complement of the target binding region of each barcoded nucleic acid molecule with (i) an oligonucleotide barcode of the plurality of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) a target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules.

10. 10. The method of any one of claims 4 to 9, comprising denaturing the plurality of extended bar-coded nucleic acid molecules prior to amplifying the plurality of extended bar-coded nucleic acid molecules.

11. 10. The method of any one of claims 7 to 9, wherein determining the copy number of the nucleic acid targets comprises determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of second molecular labels having distinct sequences associated with single-labeled nucleic acid molecules among a plurality of single-labeled nucleic acid molecules comprising the sequences of each of the plurality of nucleic acid targets.

12. 12. The method of Claim 11, wherein determining the copy number of the nucleic acid targets comprises determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of first molecular labels having distinct sequences associated with single-labeled nucleic acid molecules among a plurality of single-labeled nucleic acid molecules comprising the sequences of each of the plurality of nucleic acid targets.

13. 13. The method of claim 12, wherein the sequence of each of the plurality of nucleic acid targets comprises a subsequence of each of the plurality of nucleic acid targets.

14. 14. The method of any one of claims 1 to 13, wherein the sequence of the nucleic acid target in the plurality of barcoded nucleic acid molecules comprises a subsequence of the nucleic acid target.

15. 15. The method of any one of claims 1 to 14, wherein the first molecular label is hybridized to the second molecular label after extending the 3' ends of the plurality of barcoded nucleic acid molecules.

16. 16. The method of any one of claims 1 to 15, wherein each elongated barcoded nucleic acid molecule comprises a first molecular label, a second molecular label, a target binding region, and a complement of the target binding region.

17. The method of any one of claims 1 to 16, wherein the complement of the target binding region is complementary to a portion of the target binding region.

18. The method of any one of claims 1 to 17, wherein the target binding region comprises a gene-specific sequence and / or a poly(dT) sequence.

19. 19. The method of any one of claims 1 to 18, wherein hybridizing the complement of the target binding region of a barcoded nucleic acid molecule with the target binding region of the barcoded nucleic acid molecule itself comprises intramolecular hybridization of the target binding region within the barcoded nucleic acid molecule with the complement of the target binding region, forming a stem-loop.

20. 20. The method of claim 19, wherein the second molecular beacon is the complement of the first molecular beacon.

21. 21. The method of any one of claims 1 to 20, wherein hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises intermolecular hybridization between the complement of the target binding region of the barcoded nucleic acid molecule and the target binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes.

22. 22. The method of claim 21, wherein the second molecular label is different from the first molecular label, and the second molecular label is not the complement of the first molecular label.

23. 23. The method of any one of claims 21-22, comprising extending a 3' end of an oligonucleotide barcode hybridized to a complement of a target binding region of a barcoded nucleic acid molecule to generate a plurality of extended barcoded nucleic acid molecules, each of which comprises a complement of a first molecular label and a second molecular label.

24. 24. The method of claim 23, wherein the sequence of the second molecular label is different from the sequence of the first molecular label, and the second molecular label is not the complement of the first molecular label.

25. 25. The method of any one of claims 1 to 24, wherein hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules comprises intermolecular hybridization between the complement of the target binding region of the barcoded nucleic acid molecule and the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules.

26. 26. The method of claim 25, wherein the sequence of the second molecular label is different from the sequence of the first molecular label, and the second molecular label is not the complement of the first molecular label.

27. 27. The method of any one of claims 1 to 26, wherein the reverse transcriptase is capable of terminal transferase activity.

28. 28. The method of any one of claims 1 to 27, wherein the template switch oligonucleotide comprises one or more 3' ribonucleotides, optionally three 3' ribonucleotides, and further wherein the 3' ribonucleotide optionally comprises a guanine.

29. 29. The method of any one of claims 1 to 28, wherein the reverse transcriptase comprises a viral reverse transcriptase, which may be murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase.

30. The method of any one of claims 1 to 29, wherein the sample comprises a single cell, optionally an immune cell, further optionally a B cell or a T cell.

31. 31. The method of any one of claims 1 to 30, wherein the sample comprises a plurality of cells, a plurality of single cells, a tissue, a tumor sample, or any combination thereof.

32. The method of any one of claims 30 to 31, wherein the single cell comprises a circulating tumor cell.

33. 33. The method of any one of claims 1 to 32, wherein each oligonucleotide barcode comprises a first universal sequence.

34. 34. The method of any one of claims 1 to 33, wherein the plurality of extended barcoded nucleic acid molecules comprises a first universal sequence and a complement of the first universal sequence.

35. 35. The method of any one of claims 6 to 34, wherein amplifying the plurality of extended bar-coded nucleic acid molecules to generate a plurality of copies of the extended bar-coded nucleic acid molecules comprises using a primer capable of hybridizing to a first universal sequence, or a complement thereof.

36. 35. The method of any one of claims 6-34, wherein amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of single-labeled nucleic acid molecules comprises using a primer capable of hybridizing to a first universal sequence, or its complement, and an amplification primer.

37. 37. The method of claim 36, wherein the amplification primers are target-specific primers, and the target-specific primers may specifically hybridize to an immune receptor, a constant region of an immune receptor, a variable region of an immune receptor, a diversity region of an immune receptor, and / or a junction between a variable region and a diversity region of an immune receptor.

38. the immune receptor is a T cell receptor (TCR) and / or a B cell receptor (BCR) receptor; The TCR may comprise a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof; The method of any one of claims 37 to 37, wherein the BCR receptor may comprise a BCR heavy chain and / or a BCR light chain.

39. 39. The method of any one of claims 1 to 38, wherein extending the 3' ends of the plurality of barcoded nucleic acid molecules comprises extending the 3' ends of the plurality of barcoded nucleic acid molecules using a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity, wherein the DNA polymerase optionally comprises Klenow fragment.

40. 40. The method of any one of claims 1 to 39, comprising obtaining sequence information of a plurality of extended barcoded nucleic acid molecules, or products thereof.

41. 41. The method of Claim 40, wherein obtaining sequence information comprises attaching sequencing adapters to the plurality of extended barcoded nucleic acid molecules, or products thereof.

42. 41. The method of claim 40, wherein obtaining sequence information comprises attaching sequencing adaptors to the plurality of single-labeled nucleic acid molecules, or products thereof.

43. The method of any one of claims 40 to 42, wherein the step of obtaining sequence information comprises the step of obtaining sequence information of a BCR light chain and a BCR heavy chain of a single cell.

44. The method of claim 43, wherein the sequence information of the BCR light chain and the BCR heavy chain includes the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the BCR light chain and / or the BCR heavy chain.

45. The method of any one of claims 43 to 44, comprising pairing the BCR light chain and BCR heavy chain of a single cell based on the obtained sequence information.

46. The method of any one of claims 43 to 45, wherein the sample comprises a plurality of single cells, and the method comprises pairing the BCR light chain and BCR heavy chain of at least 50% of the single cells based on the obtained sequence information.

47. 47. The method of any one of claims 40 to 46, wherein obtaining sequence information comprises obtaining sequence information of the TCR alpha chain and the TCR beta chain of a single cell.

48. 48. The method of claim 47, wherein the sequence information of the TCR alpha chain and the TCR beta chain includes the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the TCR alpha chain and / or the TCR beta chain.

49. 49. The method of any one of claims 47 to 48, comprising pairing the TCR alpha chain and TCR beta chain of a single cell based on the sequence information obtained.

50. 50. The method of any one of claims 47 to 49, wherein the sample comprises a plurality of single cells, and the method comprises pairing the TCR alpha chain and TCR beta chain of at least 50% of the single cells based on the obtained sequence information.

51. 51. The method of any one of claims 40 to 50, wherein obtaining sequence information comprises obtaining sequence information of a TCR gamma chain and a TCR delta chain of a single cell.

52. 52. The method of claim 51, wherein the sequence information of the TCR gamma chain and the TCR delta chain comprises the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the TCR gamma chain and / or the TCR delta chain.

53. 53. The method of any one of claims 51 to 52, comprising pairing the TCR gamma chain and TCR delta chain of a single cell based on the obtained sequence information.

54. 54. The method of any one of claims 51 to 53, wherein the sample comprises a plurality of single cells, and the method comprises pairing the TCR gamma chain and TCR delta chain of at least 50% of the single cells based on the obtained sequence information.

55. 55. The method of any one of claims 1 to 54, wherein the complement of the target binding region comprises the reverse complement of the target binding region and / or the complement of the target binding region.

56. 56. The method of any one of claims 1 to 55, wherein the complement of the molecular beacon comprises the reverse complement of the molecular beacon and / or the complement of the molecular beacon.

57. 57. The method of any one of claims 1 to 56, wherein the plurality of barcoded nucleic acid molecules comprises barcoded deoxyribonucleic acid (DNA) molecules and / or barcoded ribonucleic acid (RNA) molecules.

58. 58. The method of any one of claims 1 to 57, wherein the nucleic acid target comprises a nucleic acid molecule, optionally ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof, and further wherein the mRNA optionally encodes an immune receptor.

59. 59. The method of any one of claims 58 to 58, wherein the nucleic acid target comprises a cellular component binding reagent.

60. 59. The method of claim 58, wherein the nucleic acid molecule is associated with a cellular component binding reagent.

61. 61. The method of claim 60, further comprising dissociating the nucleic acid molecule from the cellular component binding reagent.

62. 62. The method of any one of claims 1-61, wherein at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences.

63. 63. The method of any one of claims 1-62, wherein each molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides.

64. 64. The method of any one of claims 1-63, wherein a plurality of oligonucleotide barcodes are associated with the solid support, wherein each of the plurality of oligonucleotide barcodes associated with the same solid support may comprise the same sample label, and further wherein each sample label of the plurality of oligonucleotide barcodes may comprise at least six nucleotides.

65. 65. The method of any one of claims 1-64, wherein each of the plurality of oligonucleotide barcodes comprises a cell label, and wherein each cell label of the plurality of oligonucleotide barcodes optionally comprises at least 6 nucleotides.

66. 66. The method of claim 65, wherein oligonucleotide barcodes associated with the same solid support comprise the same cell label.

67. 66. The method of claim 65, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell labels.

68. 68. The method of any one of claims 65-67, wherein each of the plurality of extended barcoded nucleic acid molecules comprises a cell marker and a complement of the cell marker, and the complement of the cell marker may comprise a reverse complement of the cell marker and / or a complementary sequence of the cell marker.

69. 69. The method of any one of claims 1-68, comprising extending a plurality of oligonucleotide barcodes hybridized to copies of a nucleic acid target in the presence of one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethylsulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof.

70. 70. The method of any one of claims 1 to 69, wherein the solid support comprises a synthetic particle or a planar surface.

71. 71. The method of any one of claims 1-70, wherein the sample comprises a single cell, and comprising associating a synthetic particle comprising a plurality of oligonucleotide barcodes with the single cell in the sample.

72. 72. The method of claim 71, comprising a step of lysing the single cells after associating the synthetic particles with the single cells, wherein lysing the single cells may comprise heating the sample, contacting the sample with a detergent, altering the pH of the sample, or any combination thereof.

73. 73. The method of any one of claims 71 to 72, wherein the synthetic particle and the single cell are in the same well.

74. 73. The method of any one of claims 71 to 72, wherein the synthetic particle and the single cell are in the same droplet.

75. 75. The method of any one of claims 71 to 74, wherein at least one of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on a synthetic particle, or at least one of the plurality of oligonucleotide barcodes is encapsulated or partially encapsulated within a synthetic particle.

76. 76. The method of any one of claims 71 to 75, wherein the synthetic particles are disintegrable.

77. The composite particles include beads, the beads being 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; 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; or Collapsible Hydrogel Particles 77. The method of any one of claims 71 to 76, optionally comprising:

78. each of the plurality of oligonucleotide barcodes comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; 78. The method of any one of claims 71-77, wherein the linker functional group and the support functional group may independently be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof.

79. a plurality of oligonucleotide barcodes, each of the plurality of oligonucleotide barcodes comprising a molecular label and a target binding region, and at least 10 of the plurality of oligonucleotide barcodes comprising different molecular label sequences; reverse transcriptase, a template switching oligonucleotide, or a portion thereof, comprising a target binding region; and DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity Includes a kit.

80. 80. The kit of claim 79, wherein the DNA polymerase comprises a Klenow fragment.

81. 81. The kit of any one of claims 79 to 80, wherein the reverse transcriptase comprises a viral reverse transcriptase, optionally murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase.

82. The kit of any one of claims 79 to 81, wherein the template switch oligonucleotide comprises one or more 3' ribonucleotides, optionally three 3' ribonucleotides, and further wherein the 3' ribonucleotide optionally comprises a guanine.

83. 83. The kit of any one of claims 79-82, comprising one or more of ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethylsulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salt, betaine, or any combination thereof.

84. 84. The kit of any one of claims 79 to 83, comprising a buffer, a cartridge, one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or a combination thereof.

85. 85. The kit of any one of claims 79 to 84, wherein the target binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof.

86. 86. The kit of any one of claims 79 to 85, wherein the oligonucleotide barcodes comprise the same sample label and / or the same cell label.

87. 87. The kit of Claim 86, wherein each sample label, cell label, and / or molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides.

88. 88. The kit of any one of claims 79 to 87, wherein at least one of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on a synthetic particle and / or at least one of the plurality of oligonucleotide barcodes is encapsulated or partially encapsulated within a synthetic particle.

89. A kit according to any one of claims 79 to 88, wherein the synthetic particles are disintegrable.

90. The composite particles include beads, the beads being 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; 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; or Collapsible Hydrogel Particles 90. The kit of any one of claims 79 to 89, which may comprise:

91. each of the plurality of oligonucleotide barcodes comprises a linker functional group; the synthetic particles comprise solid support functional groups; 91. The kit of any one of claims 79-90, wherein the support functional group and the linker functional group are associated with one another, and the linker functional group and the support functional group may independently be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof.

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