Determination of 5' transcript sequence

Abstract

The present disclosure includes systems, methods, compositions, and kits for labeling nucleic acid targets in samples. In some embodiments, nucleic acid targets (e.g., mRNA) are first barcoded at the 3' end, then barcoded at the 5' end, followed by template switching reaction and inter- and / or intra-molecular hybridization and extension. In some embodiments, methods for 5'-based and 3'-based gene expression profiling are provided. In some embodiments, methods for immune repertoire profiling are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC §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 a sequence listing in electronic format. The sequence listing has been submitted as a file entitled 68EB_298703_WO, which is 4.0 kilobytes in size and was 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 specifically to multi-omics analysis using molecular barcoding. [Background technology]

[0002] Molecular barcoding methods and techniques are useful for single-cell transcriptomics analysis, such as, for example, using reverse transcription, polymerase chain reaction (PCR) amplification, and next-generation sequencing (NGS) to decipher gene expression profiles to determine the state of a cell. 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 either 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 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 of which comprises 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 isolating the complement of the target binding region of each barcoded nucleic acid molecule by (i) replicating the sequence of the target binding region of the first molecular label, a target binding region of the second molecular label, a ... the number of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) hybridizing to a target binding region of a different barcoded nucleic acid molecule of 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 number of copies 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 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 includes 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 includes 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 generate 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.

[0006] 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 of which comprises 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 coupling the complement of the target binding region of each of the barcoded nucleic acid molecules to (i) the plurality of oligonucleotide barcodes. the nucleic acid molecule is a barcode having a first molecular label and a second molecular label; and a 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule.

[0007] In some embodiments, the method includes 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. 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 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. 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 with distinct sequences associated with the single-labeled nucleic acid molecules of the plurality of single-labeled nucleic acid molecules that include 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 the single-labeled nucleic acid molecules among the plurality of single-labeled nucleic acid molecules that include the sequence of each of the plurality of nucleic acid targets. In some embodiments, the sequence of each of the plurality of nucleic acid targets comprises a subsequence of each of the plurality of nucleic acid targets. In some embodiments, the sequence of the nucleic acid target in the plurality of barcoded nucleic acid molecules comprises a subsequence of the nucleic acid target.

[0008] In some embodiments, the first molecular label is hybridized to the second molecular label after extending the 3' end of the 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 includes 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. 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 a 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 primer specifically hybridizes to the immune receptor. In some embodiments, the target-specific primer specifically hybridizes to the constant region of the immune receptor. In some embodiments, the target-specific primer specifically hybridizes to the variable region of the immune receptor. In some embodiments, the target-specific primer specifically hybridizes to the diversity region of the immune receptor. In some embodiments, the target-specific primer specifically hybridizes to the junction of the variable region and the diversity region of the immune receptor. In some embodiments, the immune receptor is a T cell receptor (TCR) and / or a B cell receptor (BCR) receptor. 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 a 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 a sequencing adaptor to the plurality of extended barcoded nucleic acid molecules, or products thereof. In some embodiments, obtaining sequence information comprises attaching a sequencing adaptor to the plurality of single-labeled nucleic acid molecules, or products thereof. In some embodiments, obtaining sequence information comprises obtaining sequence information of the BCR light chain and the BCR heavy chain of the single cell. In some embodiments, the sequence information of the BCR light chain and the BCR heavy chain comprises the sequence of the 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 comprises pairing the BCR light chain and the BCR heavy chain of the single cell based on the sequence information obtained. In some embodiments, the sample comprises a plurality of single cells, and the method comprises pairing the BCR light chain and the BCR heavy chain of at least 50% of the single cells based on the sequence information obtained. In some embodiments, the step of obtaining the sequence information comprises obtaining the 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 comprises the sequence of the 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 comprises pairing the TCR alpha chain and the TCR beta chain of the single cell based on the sequence information obtained.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 sequence information obtained. 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 the 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 sequence information obtained. 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 sequence information obtained.

[0013] In some embodiments, the complement of the target binding region comprises a reverse complement of the target binding region. In some embodiments, the complement of the target binding region comprises a complementary sequence of the target binding region. In some embodiments, the complement of the molecular label comprises a reverse complement of the molecular label. In some embodiments, the complement of the molecular label comprises a complementary sequence of the molecular label. In some embodiments, the plurality of barcoded nucleic acid molecules comprises 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, the plurality of oligonucleotide barcodes are associated with a solid support. In some embodiments, the 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 6 nucleotides. In some embodiments, the plurality of oligonucleotide barcodes each comprise a cell label. In some embodiments, each cell label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides. In some embodiments, the oligonucleotide barcodes associated with the same solid support comprise the same cell label. In some embodiments, the oligonucleotide barcodes associated with different solid supports comprise different cell labels. In some embodiments, each of the plurality of extended barcoded nucleic acid molecules comprises a cell label and a complement of the cell label. In some embodiments, the complement of the cell label comprises a reverse complement sequence of the cell label. In some embodiments, the complement of the cell label comprises a complementary sequence of the cell label. In some embodiments, the method includes 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 salts, betaine, or any combination thereof. In some embodiments, the solid support includes synthetic particles. In some embodiments, the solid support includes a planar surface.

[0015] In some embodiments, the sample includes a single cell, and the method includes associating a synthetic particle 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 particle with the single cell. In some embodiments, lysing the single cell includes heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof. In some embodiments, the synthetic particle and the single cell are in the same well. In some embodiments, the synthetic particle 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 disintegrable. In some embodiments, the synthetic particle includes a bead. In some embodiments, the beads 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. In some embodiments, the synthetic particles include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof. In some embodiments, the synthetic particles include 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 disclosure of the present specification includes a kit. In some embodiments, the kit includes a plurality of oligonucleotide barcodes, each of the plurality of oligonucleotide barcodes includes a molecular label and a target binding region, and at least 10 of the plurality of oligonucleotide barcodes include different molecular label sequences; a reverse transcriptase; a template switching oligonucleotide including a target binding region, or a portion thereof; and a DNA polymerase that lacks 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 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 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 kit comprises 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.

[0018] In some embodiments, the kit comprises a buffer. In some embodiments, the kit comprises a cartridge. In some embodiments, the kit comprises one or more reagents for a reverse transcription reaction. In some embodiments, the kit comprises 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. In some embodiments, the oligonucleotide barcodes comprise the same sample label and / or the same cell label. In some embodiments, each sample label and / or cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides. In some embodiments, each molecular label of the plurality of oligonucleotide barcodes comprises 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 disintegrable. In some embodiments, the synthetic particle comprises 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, 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 amines, aldehydes, ketones, and any combination thereof. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 illustrates a non-limiting exemplary barcode. [Diagram 2] FIG. 1 illustrates a non-limiting exemplary workflow of barcoding and electronic counting. [Diagram 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-4B] 1 shows a schematic diagram of a non-limiting exemplary method for gene-specific labeling of a nucleic acid target at the 5′ end. [Figure 5A-5B] 1 shows a schematic diagram of a non-limiting exemplary method for labeling nucleic acid targets at the 5′ end for whole transcriptome analysis. [Figures 6A-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 profile protocol (e.g., the BD Rhapsody targeted protocol) as one workflow. [Figure 8A-8C] Figure 1 shows non-limiting exemplary experimental results of capturing and sequencing 5' T cell receptor (TCR) V(D)J regions using the V(D)J protocol. The V(D)J hairpin protocol can include 3' and 5' amplification performed on the same bead using captured single, resting peripheral blood mononuclear cell (PBMC) derived mRNA molecules. The TCR alpha chain / beta chain pairing efficiency was 37.9%, which was comparable to other platforms such as Clonetech's scTCR profiling kit. [Figure 9A-9B] Non-limiting exemplary plots are shown showing improved 5'V(D)J detection sensitivity using improved V(D)J protocols. Ethylene glycol was added to aid in the reduction of secondary structure in reverse transcription (RT). Hybridization time, buffer, and template switching (TS) oligo dT length were altered 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-10E] Figure 10 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, the 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. [Figures 11A-11F]11A, 11C, 11E) and 5' amplification (FIGS. 11B, 11D, 11F). [Fig. 12A-12F] 12A, 12C, 12E) and 5' amplification (FIGS. 12B, 12D, 12F). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, similar symbols typically identify similar components unless otherwise indicated by context. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments can be used and other changes can be made without departing from the spirit or scope of the subject matter presented herein. It is readily understood that the aspects of the present disclosure, generally as described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form part of the disclosure of this specification. 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 genes expressed in cells at different developmental stages or under different environmental conditions. However, it can also be very difficult to determine the absolute number of nucleic acid molecules (e.g., mRNA molecules), especially when the number of molecules is very small. One method to determine 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, which varies with PCR cycles and gene sequences, resulting in amplification bias and inaccurate gene expression measurements. Stochastic barcodes with unique molecular labels (also called molecular indexes (MI)) 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 (ML) to label mRNA during PCR and reverse transcription (RT).

[0022] The Precise™ assay utilizes a non-exhaustive pool of stochastic barcodes with a large number, e.g., 6561-65536, of unique molecular label sequences 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 may hybridize to a stochastic barcode, thereby generating a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules obtained from the microwells of the microwell plate may be pooled into a single tube for PCR amplification and sequencing. The raw sequencing data may be analyzed to obtain the number of reads, the number of stochastic barcodes with unique molecular label 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 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 of which comprises 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 isolating the complement of the target binding region of each barcoded nucleic acid molecule by (i) replicating the sequence of the target binding region of the first molecular label, a target binding region of the second molecular label, a ... the number of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) hybridizing to a target binding region of a different barcoded nucleic acid molecule of 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 number of copies 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 contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each of which comprises 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 coupling the complement of the target binding region of each of the barcoded nucleic acid molecules to (i) the plurality of oligonucleotide barcodes. the nucleic acid molecule is a barcode having a first molecular label and a second molecular label; and a 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule.

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

[0027] definition Unless otherwise defined, 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 placed at the 5' or 3' end of the nucleic acid. When the adapter includes a known sequence at the 5' and 3' ends, the known sequence may be the same or different sequences. The adapters placed at the 5' and / or 3' ends of the polynucleotide may be capable of hybridizing to one or more oligonucleotides immobilized on a surface. The adapter may include a universal sequence in some embodiments. 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' adaptor may comprise the same and / or universal nucleic acid sequence, and the 3' adaptor may comprise the same and / or universal sequence. The universal sequence may be present in different members of a plurality of nucleic acid molecules, allowing for the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair), or more universal sequences may be present in different members of a collection of nucleic acid molecules, allowing for the replication or amplification of multiple different sequences using at least one, two (e.g., a pair), or more single universal primers that are complementary to the universal sequence. Thus, a universal primer includes a sequence that can hybridize to such a universal sequence. A molecule having a target nucleic acid sequence may be modified to add a universal adaptor (e.g., a non-target nucleic acid sequence) to one or both ends of the 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" may mean that two or more species are identifiable as being located together at a time. Association may mean that two or more species are or were in similar containers. Association may be an informational association. For example, digital information about two or more species may be stored and used to determine that one or more of the species were located together at a time. Association may 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 may refer to a covalent or non-covalent means for attaching a label to a solid or semi-solid support such as a bead. Association may be a covalent bond between a target and a label. Association may include hybridization between two molecules (e.g., a target molecule and a label).

[0030] As used herein, the term "complementary" may refer to the ability for exact pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid can hydrogen bond with a nucleotide of 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 may be "partial," where only some of the nucleotides bind, or may be complete, where there is total complementarity between the single-stranded molecules. A first nucleotide sequence may 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 may 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 may 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 probabilistic in nature, converts the problem of molecular counting into one of locating and identifying identical molecules into a series of yes / no digital questions regarding the detection of a predefined set of labels. 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 fully or partially amplifiable label. The label may be a fully 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 include a junction of a nucleic acid sequence, for example, a junction of a naturally occurring and a 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 may 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-depleting reservoir" may refer to a pool of barcodes (e.g., stochastic barcodes) that are composed of a number of different labels. A non-depleting reservoir may contain a number of different barcodes, such that when the non-depleting reservoir is associated with a pool of targets, each target is more likely to be associated with a unique barcode. The uniqueness of each labeled target molecule can be determined by random selection statistics and depends on the number of copies of the same target molecule 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 barcodes detected then allows for the 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 one 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 be present 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 comprise 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, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0034] The nucleic acid may include one or more modifications (e.g., base modifications, backbone modifications) to result in a nucleic acid with new or enhanced properties (e.g., improved stability). The nucleic acid may include a nucleic acid affinity tag. The nucleoside may be a base-sugar combination. The base portion of the nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. The nucleotide may be a nucleoside further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides that include a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl portion of the sugar. In forming a nucleic acid, 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 fold in such a manner that a fully or partially double-stranded compound results. 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, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs, and those with reverse polarity, where one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages.

[0036] Nucleic acids may 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 CH2 component moieties.

[0037] Nucleic acids may include nucleic acid mimetics. The term "mimetics" may be 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, and 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 a PNA, the sugar backbone of a polynucleotide may be replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleotides may be retained and are directly or indirectly bound 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 bound to the aza nitrogen atoms of the amide portion of the backbone. Nucleic acid may comprise a morpholino backbone structure. For example, nucleic acid may comprise a 6-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidates or other non-phosphodiester internucleoside linkages may replace phosphodiester linkages.

[0038] Nucleic acids may include linked morpholino units (e.g., morpholino nucleic acids) having heterocyclic bases attached to the morpholino ring. Linking groups may link the morpholino monomer units in the morpholino nucleic acid. Non-ionic morpholino-based oligomeric compounds may have less undesirable interactions with intracellular proteins. Morpholino-based polynucleotides may be non-ionic mimics of nucleic acids. Various compounds within the morpholino class may be linked using different linking groups. A further class of polynucleotide mimics may be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules may be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers may be prepared and used in the synthesis of oligomeric compounds using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid strands may increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to the natural complexes. Further modifications can 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 properties.

[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 bases, 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), pyridoindol 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 a plurality of barcodes (e.g., stochastic barcodes) may 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 a nucleic acid may be immobilized (e.g., covalently or non-covalently). A solid support may include 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 shape. A bead may be of a non-spherical 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 "beads."

[0041] As used herein, the term "stochastic barcode" may refer to a polynucleotide sequence that includes 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 in a sample. A stochastic barcode may be used to control errors that may occur after a label is associated 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 target binding region that is gene-specific. 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 in 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. As used herein, the term "stochastic barcoding" may refer to random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a Poisson recursive 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 may 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" may refer to a group of enzymes that have reverse transcriptase activity (i.e., catalyze the synthesis of DNA from an RNA template). In general, 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 Lactococcus lactis LI.LtrB intron reverse transcriptase, Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases can 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 can 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, more than one universal adapter sequence 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 includes the target-specific sequence and the other of which includes the barcode. For example, the universal adapter sequence can be part of an oligonucleotide that includes a target-specific sequence to generate a nucleotide sequence that is complementary to the target nucleic acid. A second oligonucleotide comprising a complementary sequence of the barcode and 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 method.

[0046] Barcode Barcoding, e.g., probabilistic barcoding, is described, for example, in US 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 entirety. In some embodiments, the barcodes disclosed herein may be probabilistic 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 number or range. 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 the stochastic barcode may be referred to as molecular labels.

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

[0048] Labels, e.g., cell labels, may include a set of unique nucleic acid subsequences of defined length, e.g., 7 nucleotides each (equivalent to the number of bits used in some Hamming error correction codes), that can be designed to provide error correction capabilities. An error correction subsequence set that includes 7 nucleotide sequences can be designed such that any pairwise combination of sequences in the set exhibits a defined "genetic distance" (or number of mismatched bases), e.g., an error correction subsequence set can be designed to exhibit a genetic distance of 3 nucleotides. In this case, consideration of the error correction sequences in the sequence data set of the labeled target nucleic acid molecule (described in more detail below) may allow amplification or sequencing errors to be detected or corrected. In some embodiments, the length of the nucleic acid subsequences used to create the error correction code may vary, e.g., 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 approximately these values ​​or such number or range of nucleotides in length. In some embodiments, nucleic acid subsequences of other lengths may be used to create the error correcting code.

[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, RNA each including 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 nucleotides or more. In some embodiments, none of the labels of the barcode are spaced by a spacer.

[0051] Universal Signage The 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 a plurality of 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 the barcode that includes the 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 the 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 for extension of the barcode or a region within the barcode. The universal label may 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 may comprise at least about 10 nucleotides. The universal label may 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 may 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 may 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 times. A dimensional label provides information about the order in which targets, groups of targets, and / or samples were barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. A cell may be pulsed again with a barcode (e.g., a stochastic barcode) in the G1 phase of the cell cycle. A cell 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, the dimensional label provides information about which targets were labeled at which stage of the cell cycle. Dimensional labels can interrogate many different biological times. Exemplary biological times can include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation.In another example, a sample (e.g., a cell, a cell population) can be stochastically labeled before and / or after treatment with a drug and / or therapy.Changes in the copy number of distinct targets can indicate the response of the sample to the drug and / or therapy.

[0053] The dimension label may be activatable. The activatable dimension label may be activated at a particular time. The activatable label may, for example, be continuously activated (e.g., not turned off). The activatable dimension label may, for example, be reversibly activatable (e.g., the activatable dimension label may be turned on and turned off). The dimension label may, for example, be reversibly activatable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The dimension label may, for example, be reversibly activatable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the dimension label may 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 a non-natural nucleotide.

[0054] The dimension label, in some embodiments, may be the same for all barcodes (e.g., stochastic barcodes) bound 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 include the same dimension label. In some embodiments, at least 60% of the barcodes on the same solid support may include the same dimension label. In some embodiments, at least 95% of the barcodes on the same solid support may include the same dimension label.

[0055] On multiple solid supports (e.g., beads), 6Many unique dimension label sequences, even more than 10, may be presented. The dimension labels may 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 about these values ​​or such number or range of nucleotides. The dimension labels 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. The dimension labels may comprise from about 5 to about 200 nucleotides. The dimension labels may comprise from about 10 to about 150 nucleotides. The dimension labels may 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 be referenced 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., a structure with an identifiable identifier, e.g., a color code, a bar code, a magnetic property, a fluorescent property, a radioactive property, or 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 code one or more locations in space.

[0057] The spatial label may be the same for all barcodes bound to a given solid support (e.g., a bead), 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 label sequences, even more than 10, may be presented. Spatial 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 about these values ​​or such number or range of nucleotides. Spatial labels 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 labels can include about 5 to about 200 nucleotides. Spatial labels can include about 10 to about 150 nucleotides. Spatial labels can include 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 to determine which target nucleic acid originates from which cell. In some embodiments, the cell label is the same for all barcodes bound to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell 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 approximately these values ​​or such number or range. In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%, or approximately these values. 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 label sequences, even more than 10, may be presented. The cell label may 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 about these values ​​or such number or range of nucleotides. The cell label may 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, the cell label may include about 5 to about 200 nucleotides. As another example, the cell label may include about 10 to about 150 nucleotides. As yet another example, the cell label may include 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 that is 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 that is hybridized to the barcode (e.g., the 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 any two of these values, or approximately these values ​​or such number or range. For example, the plurality of barcodes may include about 6561 barcode sequences having distinct sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences having distinct sequences. In some embodiments, there may be at least or at most 10 unique molecular label sequences. 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 one unique barcode sequence. 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 be different 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 about 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. 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 long.

[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 a particular type of target nucleic acid species hybridized to the barcode. A molecular label includes a nucleic acid sequence that provides a counter for the particular 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., a bead). 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 about 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 about 10 unique molecular label sequences, or a number or range between any two of these values. 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 one unique molecular label sequence. A barcode with a unique molecular label sequence may 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 label 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 more. 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 that includes 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 to 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, a 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 independent 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 a 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 length or greater multimer sequence. 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. A target binding region can be 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 about these values ​​or such number or range of nucleotides in length. A target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or more.

[0068] In some embodiments, the target binding region can comprise an oligo(dT) that can hybridize with mRNA that comprises polyadenylated ends. The target binding region can be gene-specific. For example, the target binding region can be configured to hybridize to 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 may 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 may be about 5-30 nucleotides in length. When the barcode comprises 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 moieties for isoelectric focusing. Different barcodes may include different isoelectric focusing points. Upon introducing these barcodes into a sample, the sample may undergo isoelectric focusing to orient the barcodes in a known manner. In this manner, the orientation properties may be used to develop a known map of the barcodes in the sample. Exemplary orientation properties may 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 of self-assembly may self-assemble into a particular orientation (e.g., a nucleic acid nanostructure) when activated.

[0070] affinity properties A barcode (e.g., a stochastic barcode) may include one or more affinity features. For example, a spatial label may include an affinity feature. An affinity feature may include a chemical and / or biological moiety that can facilitate binding of the barcode to another entity (e.g., a cellular receptor). For example, an affinity feature may include an antibody, e.g., an antibody specific to a particular moiety (e.g., a receptor) on a sample. In some embodiments, an antibody may direct a barcode to a particular cell type or molecule. Targets at and / or near a particular cell type or molecule may be labeled (e.g., stochastically labeled). An affinity feature may provide spatial information in addition to the nucleotide sequence of the spatial label, in some embodiments, since an antibody may direct a barcode to a particular location. An antibody may be a therapeutic antibody, e.g., a monoclonal or polyclonal antibody. An antibody may be humanized or chimeric. An antibody may 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 a full-length antibody. Antibody fragments can include isolated fragments of the variable regions of an antibody, such as "Fv" fragments 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 may include one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, may include a universal adapter primer. The universal adapter primer may refer to a nucleotide sequence that is universal across all barcodes. The universal adapter primer can be used to construct a gene-specific barcode. The universal adapter primer may 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 in length. 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 between 5 and 30 nucleotides in length.

[0073] Linker When the 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 The barcodes disclosed herein, e.g., stochastic barcodes, may be associated with a solid support in some embodiments. 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 5-20 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, ceramic, plastic, glass, methylstyrene, acrylic polymer, 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 include polymer-based gels. The gel beads can be generated, for example, by encapsulating one or more polymer precursors in a droplet. The gel beads can be generated when the polymer precursor is exposed to an accelerant (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, for example, under desired conditions. The desired conditions may include environmental conditions. The desired conditions may result in the dissolution, melt, or decomposition of the polymer beads in a controlled manner. Gel beads may dissolve, melt, or decompose due to chemical, physical, biological, thermal, magnetic, electrical, or optical stimuli, or any combination thereof.

[0078] Analytes and / or reagents, e.g., oligonucleotide barcodes, may be linked / immobilized, for example, to the interior surface of the 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 the 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, linkage / immobilization of reagents to gel beads or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical crosslinker, including chemical crosslinkers described herein). Upon application of a stimulus, the labile moiety may be cleaved and the immobilized reagent released. In some embodiments, the labile moiety is a disulfide bond. For example, in cases where oligonucleotide barcodes are immobilized to gel beads via disulfide bonds, exposing the disulfide bond to a reducing agent may 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 that links 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(diallyldimethyl-ammonium 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 the collapse, dissolution, or disintegration of beads. Examples of these chemical changes can include, but are not limited to, pH-mediated changes to the bead wall, collapse of the bead wall via chemical cleavage of cross-links, triggering depolymerization of the bead wall, and switching reactions of the bead wall. Bulk changes can also be used to trigger the collapse of the beads.

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

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

[0083] Beads can also be induced to release their contents upon application of a thermal stimulus. Temperature changes can cause various changes in beads. Heat changes can cause the beads to melt, such that the bead walls collapse. In other cases, heat can increase the internal pressure of the bead's internal components, such that the bead collapses or explodes. In yet other cases, heat can transform the bead into a compressed, dehydrated state. Heat can also act on heat-sensitive polymers within the bead's walls, causing the bead to collapse. The inclusion of magnetic nanoparticles in the bead walls of microcapsules can trigger the collapse of beads as well as guide the beads in arrays.The device of the present disclosure can include magnetic beads for any purpose.In one example, the incorporation of Fe3O4 nanoparticles into polyelectrolyte-containing beads triggers collapse in the presence of an oscillating magnetic field stimulus. Beads can also disintegrate, dissolve, or break down as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrosensitive beads can trigger both disintegration of beads 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 so that the release of internal reagents can be controlled. In another example, the 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 light triggers are possible, including systems that use various molecules such as nanoparticles and chromophores that can absorb photons of a specific range of 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 walls. In yet another example, photoswitchable materials, such as azobenzene groups, can be incorporated into the bead walls. 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 light switches results in bead walls 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 a plurality of microwells of a microwell array in block 208, beads can be introduced into a plurality of 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 include a barcode sequence selected from a group including at least 100 or 1000 barcode sequences, each having a unique sequence. In some embodiments, different barcodes associated with a solid support may include barcodes with different sequences. In some embodiments, a percentage of the barcodes associated with a solid support include the same cell label. 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 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, the barcodes associated with the solid supports may have the same cell marker. The barcodes associated with different solid supports may have different cell markers selected from a group including at least 100 or 1000 cell markers 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 the multiple labels in the sample may be performed using a solid support comprising multiple synthetic particles associated with multiple barcodes. In some embodiments, the solid support may comprise multiple synthetic particles associated with multiple barcodes. The spatial labeling of the multiple barcodes on different solid supports may differ by at least one nucleotide. The solid support may comprise multiple 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, matrix, hydrogel, needle array device, 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 the 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. A bead may include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar configurations that can immobilize nucleic acids (e.g., covalently or non-covalently). A bead may be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. A bead may be or include discrete particles that are 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. In some embodiments, a 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 labels are 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 the 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 be different 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 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 9In some embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 200, no more than 300, no more than 400, no more than 500, no more than 600, no more than 700, no more than 800, no more than 900, no more than 1000, or more, of the plurality of particles comprise oligonucleotides having the same cellular sequence. In some embodiments, the plurality of particles comprising oligonucleotides with the same cellular sequence may be 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. In some embodiments, none of the plurality of particles have the same cellular labeling sequence.

[0090] The oligonucleotides on each particle may include different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences may be 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 pcs or 10 9 For example, at least 100 of the plurality of oligonucleotides comprise different barcode sequences. As another example, 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 in a single particle comprise different barcode sequences. Some embodiments provide a plurality of particles comprising barcodes. In some embodiments, the ratio of occurrence (or copies or numbers) of targets to be labeled to 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 about these values ​​or such numbers or ranges.

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

[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 the barcode on the beads, which can serve as a positional address. Examples of beads may 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 or more 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 may comprise an insoluble, semi-soluble, or insoluble material. A solid support may be referred to as "functionalized" if it comprises a linker, scaffold, building block, or other reactive moiety attached thereto, but may be "non-functionalized" if it lacks such reactive moieties attached thereto. A solid support may be freely used in solution, e.g., in a microtiter well format, in a flow-through format, e.g., 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, a microparticle, a nanoparticle, a plate, an array, a caliper, a flat support, such as a glass fiber filter, a glass surface, a metal surface, a metal surface (steel, gold silver, aluminum, silicon, and copper), a glass support, a plastic support, a silicon support, a chip, a filter, a membrane, a microwell plate, a slide, a multi-well plate or a plastic material (e.g., made of polyethylene, polypropylene, polyamide, polyvinylidene difluoride) including a membrane, and / or a wafer, a comb, a pin, or a 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 with or without a filter bottom. The solid support may include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix may be capable of penetrating intracellular spaces (e.g., around organelles). The polymer matrix may 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, for example, include a plurality of microwells. For example, a substrate may be a well array that includes 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., 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 a plurality of targets in the sample. Mapping the plurality of 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., stochastic barcoding) the plurality of targets in the sample. Visualizing a plurality of targets in the sample may include mapping the plurality of targets to a map of the sample. Mapping the plurality of 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 a sample.In some embodiments, two-dimensional and three-dimensional maps can be created before or after dissolving the sample.Dissolving the sample before or after creating the two-dimensional or three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0099] In some embodiments, barcoding the multiple targets comprises hybridizing a plurality of barcodes to the multiple targets to generate barcoded targets (e.g., stochastically barcoded targets). Barcoding the multiple 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 the cells 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 separate target can associate with a separate 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 partitioning of the cells and barcodes, 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, mechanical, or optical lysis. Cells can 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 of the target with the barcode, the diffusion rate of the target molecule can be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate. In some embodiments, the sample may be lysed by using filter paper, which can be soaked with a lysis buffer over the filter paper, and pressure can be applied to the sample that can 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, heat 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 the addition of 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 include 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 include a detergent (e.g., SDS, Li dodecyl sulfate, triton X, tween, NP-40). The detergent concentration 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 detergent in the dissolution 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 detergent in the dissolution buffer is about 1% Li-dodecyl sulfate.The time used in the method for dissolution can depend on the amount of detergent used.In some embodiments, the more detergent used, the less time is required for lysis. The lysis buffer may include 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 include a reducing agent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing agent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM, or higher. The concentration of the reducing agent 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 agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may include 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] Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysis can be performed for about 1, 5, 10, 15, 20 minutes, or longer. Lysed cells may 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 may 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] Binding of barcodes to target nucleic acid molecules After lysis of the cells and release of the nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with the barcodes of the co-localized solid support. The association may include 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). The assay conditions (e.g., pH of the buffer, ionic strength, temperature, etc.) used for hybridization may be selected to promote the formation of specific stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells may associate with multiple probes on the substrate (e.g., hybridize with the probes on the substrate). If the probes include oligo(dT), the 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 the cDNA molecules. For example, in the non-limiting example of barcoding shown in block 216 of FIG. 2, the mRNA molecules may hybridize to the barcodes on the beads. For example, the single-stranded nucleotide fragment may 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 that may allow 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 may then be ligated to any nucleic acid molecule that includes a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) may be used to connect the two fragments. For example, in a non-limiting example of barcoding shown in block 220 of Figure 2, the labeled targets (e.g., target-barcode molecules) from multiple cells (or multiple samples) can then be pooled, e.g., in a tube. The labeled targets can be pooled, e.g., by collecting beads to which barcodes and / or target-barcode molecules are attached. The recovery of the solid support-based collection of bound target-barcode molecules can be achieved by the use of magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can proceed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Further processing reactions can be performed in microwells, i.e., without first pooling the labeled target nucleic acid molecules from multiple cells.

[0106] Reverse transcription The present disclosure provides a method for creating a target-barcode conjugate using reverse transcription (e.g., block 224 of FIG. 2). The target-barcode conjugate may 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 may occur by adding a reverse transcription primer along with a reverse transcriptase. The reverse transcription primer may be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo(dT) primer may be 12-18 nucleotides in length, or may be about 12-18 nucleotides in length, and binds to an endogenous poly(A) tail at the 3' end of a mammalian mRNA. The random hexanucleotide primer may 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 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. Generally, oligo(dT) primers are 12-18 nucleotides in length and bind to endogenous poly(A) tails 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 multiplex 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 that includes 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 sequence in vitro by simultaneous primer extension of complementary strands of DNA.As used herein, PCR can include 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] The amplification of the labeled nucleic acid may include non-PCR-based methods. 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), 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 double strand is cleaved before extension reaction 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, the amplification does not generate 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 include 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 include 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 include DNA, RNA, or a combination thereof. The nucleic acid of the present disclosure may include a synthetic or modified nucleic acid.

[0112] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile or triggering nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acid (PNA), morpholino nucleic acid and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). 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 as specific cycles or time points of the amplification reaction.

[0113] Conducting one or more amplification reactions may include the use of one or more primers. The one or more primers may, for example, comprise 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 comprise 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 comprise less 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 constant 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 the 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 include a subset of all nucleic acids in one or more samples. The targets may include 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 put the PCR product into an Illumina sequencing library. Sequencing using 150bp x 2 sequencing can reveal cell label and barcode sequence (e.g., molecular label) on read 1, gene on read 2, and sample index 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 the solid support. For example, an enzyme can be used to remove nucleic acids from a substrate. For example, a nucleic acid can be removed from a substrate by restriction endonuclease digestion. For example, a nucleic acid containing dUTP or ddUTP can be treated with uracil-d-glycosylase (UDG) to remove nucleic acids from a substrate. For example, an enzyme that performs nucleotide excision, e.g., a base excision repair enzyme, e.g., an apurinic / apyrimidinic (AP) endonuclease, can be used to remove nucleic acids from a substrate. In some embodiments, a photocleavable group 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 include 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 it is synthesized (e.g., reverse transcribed). Amplification can be performed in a multiplex 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, for example, using bridge amplification. To generate ends compatible with bridge amplification using oligo(dT) probes on the substrate, a homopolymer tail can be added to the cDNA. In bridge amplification, the primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to the solid particle. When a sample containing the template nucleic acid is contacted with the particle and one thermal cycle is performed, the template molecule can be annealed 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. In the extension step, the second primer can be extended in the opposite direction by adding nucleotides in 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, one end of which is bound to the particle surface through the first and second primers, respectively, and the other end of which is unbound, respectively. In the annealing and extension step of this second cycle, each strand can hybridize to a further 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 may 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. The amplification of the labeled nucleic acid may include PCR-based or non-PCR-based methods. The amplification of the labeled nucleic acid may include exponential amplification of the labeled nucleic acid. The amplification of the labeled nucleic acid may include linear amplification of the labeled nucleic acid. The amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivatives 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, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which primers are hybridized to nucleic acid sequences and the resulting double strand is cleaved before extension reaction and amplification, strand displacement amplification using nucleic acid polymerases that lack 5' exonuclease activity, rolling circle amplification, and / or branch extension amplification (RAM).

[0120] In some embodiments, the method disclosed herein further comprises 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 or modified nucleic acid.

[0121] In some embodiments, the method includes repeatedly amplifying the labeled nucleic acid to generate multiple amplicons. The method 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 the one or more samples that include 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 acid (PNA), morpholino nucleic acid and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Non-natural nucleotides can be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify products as specific cycles or time points of the amplification reaction.

[0123] The one or more amplification reactions may include the use of one or more primers. The one or more primers may include one or more oligonucleotides. The one or more oligonucleotides may include at least about 7-9 nucleotides. The one or more oligonucleotides may include less than 12-15 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 constant 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 acid may include a subset of the total nucleic acids in one or more samples. In some embodiments, the primer is a probe 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 comprises generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of those targets. The barcode sequences of the different barcodes (e.g., molecular labels of different stochastic barcodes) may be different from each other. Generating an indexed library of barcoded targets comprises generating a plurality of indexed polynucleotides from the plurality of targets in a sample. For example, for an indexed library of barcoded targets including a first indexed target and a second indexed target, the label region of the first indexed polynucleotide may differ from the label 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 any two of these values, or by about these values ​​or such number or range, or by at least these values ​​or such number or range, or by up to these values ​​or such number or range. 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 of which comprises a cDNA region and a label region, where 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) may 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 perform PCR amplification bias minimization. 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 of 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 with 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 a labeled cDNA molecule 304 that includes a cDNA region 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 3-20 nucleotides. In some embodiments, the molecular label sequence may comprise 3-20 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 3-20 nucleotides. In some embodiments, the cell label comprises 3-20 nucleotides.

[0128] In some embodiments, the label region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, the label region 314 may include one or more of a universal label, a dimensional label, and a cell label. The 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 between any of these values, or may be approximately these values ​​or such number or range of nucleotides in length, or may be at least these values ​​or such number or range of nucleotides in length, or may be up to these values ​​or such number or range of nucleotides in length. 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 between any of these values, or may be approximately, or may be at least, or may be up to, these values ​​or such numbers or ranges 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 between any of these values, or may be approximately, or may be at least, or may be up to, these values ​​or such numbers or ranges 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 between any of these values, in length, or may be approximately these values ​​or such number or range of nucleotides in length, or may be at least these values ​​or such number or range of nucleotides in length, or may be up to these values ​​or such number or range 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., a barcode sequence or molecular label 318 and a cell label 320, or may include approximately these values ​​or such number or range of different labels, e.g., a barcode sequence or molecular label 318 and a cell label 320, or may include at least these values ​​or such number or range of different labels, e.g., a barcode sequence or molecular label 318 and a cell label 320, or may include up to these values ​​or such number or range of different labels, e.g., a barcode sequence or molecular label 318 and a 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 these values ​​or such number or range of nucleotides in length, or may be at least these values ​​or such number or range of nucleotides in length, or may be up to these values ​​or such number or range 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 between any of these values, or about these values, or at least these values, or at most these values, or up to ...

[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 using 10, 20, 40, 50, 70, 80, 90, ... 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 , or any of these values, or between these values, or about these values, or at least these values, or at most these values. 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 FIG. 3, the products from the PCR amplification in step 2 may 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 amplicons 322 can be further amplified by nested PCR. Nested PCR may 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. The 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 of different nested PCR primers 330 between any of these values, or may contain about these values ​​or such number or range of different nested PCR primers 330, or may contain at least these values ​​or such number or range of different nested PCR primers 330, or may contain up to these values ​​or such number or range of different nested PCR primers 330. The nested PCR primers 332 may contain an adaptor 334 and hybridize to a region within the cDNA portion 306'' of the labeled amplicon 322. Universal primer 328' contains adaptor 336 and can hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 generates adaptor-labeled amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328' may not contain adaptors 334 and 336. Instead, adaptors 334 and 336 can ligate to the product of nested PCR to generate adaptor-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, which allows 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 disclosure herein includes systems, methods, compositions, and kits for attaching barcodes (e.g., stochastic barcodes) having molecular labels (or molecular indexes) to the 5' end 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 supplement or complement, for example, 3'-based transcript counting methods (e.g., Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ), 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, substitutions. In some embodiments, the method can involve intramolecular hybridization.

[0134] 4A-4B show schematic diagrams of non-limiting exemplary methods 400 for gene-specifically labeling nucleic acid targets at the 5' end. A barcode 420 (e.g., a stochastic barcode) containing a target binding region (e.g., a poly(dT) tail 422) may 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 may 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 sequences of molecular labels in the barcodes for each sample may 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 may be selective, terminal transcriptase and deoxyadenosine triphosphate (dATP) can be added in block 406 to promote 3' poly(dA) tailing to generate 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 at the end of the poly(dT) region 422 to form 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 that includes a barcode at the 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 schematic diagrams of a non-limiting exemplary method 500 of labeling a nucleic acid target at the 5' end for whole transcriptome analysis. A barcode 420 (e.g., a stochastic barcode) that includes 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, a barcode 420 that includes a target binding region can bind to a nucleic acid target for labeling or barcoding. The barcode 420 can include 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 regions 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), respectively, and to track the sample origin of transcripts 424. 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 molecule 434 in block 402, terminal transferase can be used for A-tailing of the 3' end (equivalent to the 5' end of the labeled RNA transcript) of barcoded cDNA molecule 434 to generate cDNA molecules 436c each including a 3' poly(dA) tail 438 in block 406. Intramolecular hybridization of cDNA molecule 436c with 3' poly(dA) tail 438 is initiated (e.g., by heating and cooling cycles or by diluting barcoded cDNA molecule 436c with poly(dA) tail 438) such that new 3' poly(dA) tail 438 can anneal with poly(dT) tail 422 of the same labeled cDNA molecule to generate hairpin or stem-loop structure 440 of barcoded cDNA molecule in block 410. In block 412, a polymerase (e.g., Klenow enzyme) can be added with dNTPs to facilitate 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 adaptors 432, 432rc or sequences (or subsequences) of adaptors 432, 432rc. In block 418, for sequencing (e.g., using an Illumina (San Diego, Calif., US) sequencer), methods such as tagmentation or random priming can be used to generate smaller fragments of amplicon 444 that contain sequencing adaptors (e.g., P5 446 and P7 448 sequences).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 disclosure herein 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 label 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 label sequences 428; extending copies of the nucleic acid target 424 hybridized to the oligonucleotide barcodes 420 to generate a plurality of nucleic acid molecules 434, each of which comprises a sequence 450c complementary to at least a portion of the nucleic acid target 424, in block 402; 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 of which includes a target binding region 422 and a complement of the target binding region 438; hybridizing the target binding region 422 to 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 a 3' end of each of the plurality of barcoded nucleic acid molecules including 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, the molecular label 428 is hybridized to the complement 428rc of the molecular label after extending the 3' end 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) prior to amplifying the plurality of extended barcoded nucleic acid molecules 442. Contacting the copies of the nucleic acid target 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 the copies of the nucleic acid target 424 may include extending the copies of the plurality of nucleic acid targets 424 hybridized to the oligonucleotide barcodes 420 to generate a plurality of barcoded nucleic acid molecules 436c, each of which includes 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 of the plurality of single-labeled nucleic acid molecules 444c that include a respective sequence 452c of the plurality of nucleic acid targets 424. The respective sequence 452c of the plurality of nucleic acid targets may include a subsequence (including a complement or a reverse complement) 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), where at least ten 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 complementary sequence of the target binding region 422. The method includes generating a plurality of barcoded nucleic acid molecules 436 comprising a complement 438 of a target binding region 422 of each of the plurality of barcoded nucleic acid molecules 436c; hybridizing (410) the target binding region 422 to 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 disclosure herein includes a method for binding an oligonucleotide barcode to a target 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 of the nucleic acid target 424, a molecular label 428, and a target binding region 422, where at least ten of the plurality of oligonucleotide barcodes 420 include different molecular label sequences 428; and binding 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 complement 438 of the target binding region 422. 22 and a complement 438 of the target binding region 422, hybridizing (410) 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 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 includes 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 target 424 includes contacting the copies of the nucleic acid target 424 with a plurality of oligonucleotide barcodes 420, each of the plurality of oligonucleotide barcodes 420 including a target binding region 422 capable of hybridizing to the nucleic acid target 424, and extending (402) the copies of the nucleic acid target 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, where 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 of which comprises 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 of which includes 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) the 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 that contain them are denatured. In some embodiments, the sequence of the nucleic acid target in the plurality of barcoded nucleic acid molecules comprises a subsequence 452c of the nucleic acid target. The target binding region may comprise a gene specific sequence. Binding (406) an oligonucleotide comprising a complement 438 of the target binding region 422 may comprise 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 a 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 a 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 that lacks at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. The DNA polymerase may include 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 a reverse complement sequence of the target binding region. The complement of the target binding region 438 may comprise a complementary sequence of the target binding region. The complement of the molecular label 428rc may comprise a reverse complement sequence of the molecular label. The complement of the molecular label may comprise a complementary sequence of the molecular label.

[0147] In some embodiments, the plurality of barcoded nucleic acid molecules 434 may include barcoded deoxyribonucleic acid (DNA) molecules. The barcoded nucleic acid molecules 434 may include barcoded ribonucleic acid (RNA) molecules. The nucleic acid target 424 may include a nucleic acid molecule. The nucleic acid molecule may include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA including 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 US 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 single-cell multi-omics information, 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 dissociating the nucleic acid molecule and 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 the target binding region in each of the plurality of barcoded nucleic acid molecules hybridizes (410) with 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 the target binding region 422 in each of the plurality of barcoded nucleic acid molecules 436c hybridizes (410) with a complement 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 the target binding region 422 in each of the plurality of barcoded nucleic acid molecules 436c hybridizes (410) with a complement of the target binding region 438 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 of which includes a molecular label 428 and a complement of the molecular label 428rc. 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 of which includes a molecular label 428 and a complement of the molecular label 428rc. 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 including a molecular label 428 and a complement 428rc of the molecular label. The solid support may include a synthetic particle 454. The solid support may include a planar or substantially planar surface (e.g., a slide such as a microscope slide or a cover slip).

[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 are hybridized (410) to form a stem loop 440. For example, such intramolecular hybridization may 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 may be in solution when the 3' end of the plurality of barcoded nucleic acid molecules is extended (412) to extend the stem loop 440 and generate a plurality of extended barcoded nucleic acid molecules 442, each of which includes 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 detergent, 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 comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particle 454 may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The synthetic particle 454 may include a collapsible hydrogel particle. Each of the plurality of oligonucleotide barcodes 420 may include a linker functional group. The synthetic particle 454 may include 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 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 kit includes a plurality of oligonucleotide barcodes 420, each of the plurality of oligonucleotide barcodes 420 including a molecular label 428 and a target binding region (e.g., poly(dT) sequence 422), and at least ten of the plurality of oligonucleotide barcodes 420 including 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 6 nucleotides. Each molecular label of the plurality of oligonucleotide barcodes may comprise at least 6 nucleotides.

[0155] In some embodiments, at least one of the plurality of oligonucleotide barcodes 420 is partially immobilized on the synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be partially immobilized on the synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be encapsulated within the synthetic particle 454. At least one of the plurality of oligonucleotide barcodes 420 may be partially encapsulated within the synthetic particle 454. The synthetic particle 454 may be disintegrable. The synthetic particle 454 may comprise beads. The beads may comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The synthetic particles 454 may include collapsible hydrogel particles. Each of the plurality of oligonucleotide barcodes may include a linker functional group. The synthetic particles 454 may include 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 the analysis of 3'-only mRNA transcript information, which may limit the 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'-end and 5'-end of the transcripts in a high-throughput manner using the BD Rhapsody platform.

[0157] The disclosed method can be used to identify the VDJ region of B cell receptor (BCR), T cell receptor (TCR) and antibody. VDJ recombination, also known as somatic recombination, is a mechanism of genetic recombination in the early stages of immune system immunoglobulin (Ig) (e.g., BCR) and T cell receptor (TCR) production. VDJ recombination can combine variable (V), diversity (D) and joining (J) gene segments in a nearly random manner. The randomness of selecting various genes allows it to code a variety of proteins that match antigens derived from bacteria, viruses, parasites, dysfunctional cells such as tumor cells, and pollen.

[0158] The VDJ region may contain a large locus of 3 Mb, including variable (V), diversity (D) and mating (J) genes. These are the segments that may participate in VDJ recombination. There may also be constant genes that may not undergo VDJ recombination. The first event in the VDJ recombination of this locus may be the rearrangement of one of the D genes to 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 later codes for the variable segment of the heavy chain protein. Both of these steps may be catalyzed by recombinase enzymes that may delete the intervening DNA. This recombination process occurs stepwise in precursor B cells, resulting in the diversity required for the antibody repertoire. Each B cell can produce only one antibody (e.g., BCR). This specificity can be achieved by allelic exclusion, such that the functional rearrangement of one allele transmits a signal to prevent 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 may refer to T cells with the same TCR. T cells may be part of a T cell line, which may include a mixed population of T cells with TCRs, T cell clones and different TCRs, but 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, splenic tissue, and tumors. T cells can be obtained from a unit of blood taken from a subject, such as using Ficoll separation. Cells derived from an individual's circulating blood can be obtained by apheresis or leukapheresis. Apheresis products may include T cells, monocytes, granulocytes, lymphocytes, including B cells, other nucleated white blood cells, 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, for example, CD28+, CD4, CDC, 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., 3×28) conjugated beads, for example, DYNABEADS® M-450 CD3 / CD28 T, or XCYTE DYNABEADS™, for a time sufficient for positive selection of desired T cells. Immune cells (e.g., T cells and B cells) can be antigen-specific (e.g., specific for tumors). 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, for example, from 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. Different subtypes of T cells can be distinguished by the expression of different 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. The TCRs can be used to define the identity of individual T cells, allowing 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 an individual, 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 about 40 kDa and can have variable and constant domains. Genes encoding alpha and beta chains can be organized in 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. Most of the predominant histocompatibility genes are known as major histocompatibility complexes (MHCs). Thus, the complex recognized by the T cell receptor consists of MHC / peptide ligand.

[0163] In some embodiments, the disclosed methods, devices, and systems can be used for sequencing and pairing of 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., well) that contains a single solid support (e.g., bead). The cell can be lysed. The bead can contain a stochastic label that can bind to a specific location in 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. The TCR alpha and beta chains that contain 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 for pairing of receptors of BCR and heavy and light chains of antibodies. The disclosed methods allow the repertoire of immune receptors and antibodies in an individual organism or cell population to be determined. The disclosed methods can help determine the pairs of polypeptide chains that make up the immune receptor. B cells and T cells each express immune receptors, B cells express immunoglobulins and BCRs, and T cells express T cell receptors (TCRs). Both types of immune receptors can contain two polypeptide chains. Immunoglobulins can contain variable heavy (VH) chains and variable light (VL) chains. There are two types of TCRs, one consisting of alpha and beta chains and one consisting of delta and gamma chains. The polypeptides in the immune receptors can contain constant and variable regions. The variable regions can result from recombination and end-joining rearrangements of gene fragments in the chromosomes of B or T cells. In B cells, further diversification of the variable regions can occur by somatic hypermutation. The immune system has a large repertoire of receptors, and any given pair of receptors expressed by lymphocytes may be encoded by a separate, unique pair of transcripts. Knowledge of the sequences of immune receptor chain pairs 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) that contains 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 that contain 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 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. In some embodiments, the method includes contacting copies of the nucleic acid target with a plurality of oligonucleotide barcodes, each of which comprises 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) detecting the complement of the target binding region of each barcoded nucleic acid molecule by a sequence detection method comprising: (a) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules; (b) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules by a sequence detection method comprising: (i) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules; and (c) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules by a sequence detection method comprising: (i) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules; and (ii) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules by a sequence detection method comprising: (i) detecting the complement of the target binding region of each of the barcoded nucleic acid molecules; the number of oligonucleotide barcodes, (ii) the barcoded nucleic acid molecule itself, and / or (iii) hybridizing to a target binding region of a different barcoded nucleic acid molecule of 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 number of copies 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 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, and 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 the number of the 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 includes determining the copy number of the nucleic acid target in the sample based on the number of the 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 generate 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 method includes contacting a copy of the nucleic acid target with a plurality of oligonucleotide barcodes, each of which comprises 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 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 coupling the complement of the target binding region of each barcoded nucleic acid molecule to (i) the plurality of oligonucleotide barcodes. the nucleic acid molecule is a barcode having a first molecular label and a second molecular label; and a 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule. The 3' end of the barcoded nucleic acid molecule is a 3' end of the barcoded nucleic acid molecule.

[0170] Some embodiments of the method 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 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. 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 a nucleic acid target may include determining the copy number of each of the plurality of nucleic acid targets in a sample based on the number of second molecular labels with distinct sequences associated with a single-labeled nucleic acid molecule of the plurality of single-labeled nucleic acid molecules that include the sequence of each 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 multiple nucleic acid targets in the sample based on the number of first molecular labels having distinct sequences associated with the single-labeled nucleic acid molecules among the multiple single-labeled nucleic acid molecules that include the sequence of each of the multiple nucleic acid targets.The sequence of each of the multiple nucleic acid targets may include a subsequence of each of the multiple nucleic acid targets.The sequence of the nucleic acid target in the multiple barcoded nucleic acid molecules may include a subsequence of the nucleic acid target.

[0171] In some embodiments, the method includes the addition (e.g., by a template switching reaction) of a complement of the target binding region to the end (e.g., 3' end) of the barcoded nucleic acid molecule. In some embodiments, the method includes 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 an amplicon thereof), 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 the 3' poly(dT) capture bead. The mRNA molecule may be captured on the bead by the 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's sequencing platform. Access to the barcoded 5' sequence can allow for the detection of splice variants and sequence diversity that occurs in the variable regions of the T cell receptor (TCR) and B cell receptor (BCR), as well as the 5' end of the transcript.

[0172] 6A-6K show schematics 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 that maintain their integrity through a wide range of physical and chemical manipulations. After poly(A) capture of the 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 allow for 3'- and / or 5'-based sequencing. The methods can allow for flexibility in sequencing. In some embodiments, the methods can allow for profiling of immune repertoires of both T and B cells 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 those transcripts can be captured in a single experiment. The methods disclosed herein can allow for V(D)J detection of both T and B cells (e.g., hypermutation).

[0174] The methods and systems described herein can be used with respect to methods and systems that use antibodies associated with (e.g., bound to or conjugated with) 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 entirety. In some embodiments, the methods disclosed herein allow for T and B cell V(D)J profiling, 3' targeting, 5' targeting, 3' whole transcriptome amplification (WTA), 5' WTA, protein expression profiling with 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 schematics 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., stochastic barcode, oligonucleotide barcode 602) may include a target binding region (e.g., poly(dT) 604) that may bind to the nucleic acid target (e.g., polyadenylated RNA transcript 606) by a poly(dA) tail 608 or to other nucleic acid targets for labeling or barcoding (e.g., unique labeling). The target binding region may include gene-specific sequences, oligo(dT) sequences, random multimers, 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 the 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 include polymer-based gels. The gel beads can be generated, for example, by encapsulating one or more polymer precursors in a droplet. The gel beads can be generated when the polymer precursors are exposed to an accelerant (e.g., tetramethylethylenediamine (TEMED)).

[0176] 6A illustrates a non-limiting exemplary embodiment of a reverse transcription reaction 600a. During reverse transcription 600a, when the end of the oligonucleotide barcode 602 is reached, the activity of a 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 the newly synthesized cDNA sequence strand 614c (the antisense sequence of the RNA sequence 614r). These CCC bases 612 can serve as anchor sites for a template switch oligonucleotide 616 (e.g., a template switching oligonucleotide) that includes a sequence complementary to the tailed sequence (e.g., rGrGrG 618). The template switch oligonucleotide 616 can include at least a portion of the target binding region 604. Upon base pairing between rGrGrG 618 and the added deoxycytidine stretch 612, the enzyme "switches" the template strand from the oligonucleotide barcode 602 to the template switch oligonucleotide 616 and continues replication to the 5' end of the template switch oligonucleotide 616. Thus, the resulting first strand labeled cDNA (e.g., barcoded nucleic acid molecule 620) contains the reverse complement sequence of the template switch oligonucleotide 616 and thus may include the complement (e.g., reverse complement) of the target binding region (e.g., poly(dA) 608). The barcoded nucleic acid molecule 620 may include cDNA 614c (the reverse complement sequence of RNA sequence 614r). The reaction may be performed in the presence of one or more additives configured to reduce secondary structure (e.g., ethylene glycol). The barcoded nucleic acid molecule 620 may also include some labels.The oligonucleotide barcodes 602 may include a first molecular label (ML1) 622 and a sample label (e.g., a compartment label, a cell label (CL) 624) for labeling the transcripts 606 and tracking the sample origin of the RNA transcripts 606 (or nucleic acid targets such as antibody oligonucleotides associated with or dissociated from the antibody), 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 the oligonucleotide barcodes for each sample may be sufficiently large for stochastic labeling of the RNA transcripts. In some embodiments, the sample label is a compartment label. In some embodiments, the sample label is a cell label. The barcoded nucleic acid molecule 620 may undergo a denaturation step 600b (e.g., a denaturing step), thereby resulting in 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 the 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" may 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 allowing the synthesis of successive product DNAs that directly link the adapter sequence to the target oligonucleotide sequence without ligation. Template switching may include adapter ligation, homopolymer tailing (e.g., polyadenylation), random primers, or oligonucleotides that polymerase 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 a reverse complement of the target binding region or may comprise a complementary sequence of the target binding region. The complement of a molecular label may comprise a reverse complement of the molecular label or may comprise a 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 comprising 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 comprise 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. Each molecular label of the plurality of oligonucleotide barcodes may comprise at least six nucleotides.

[0181] In some embodiments, the plurality of oligonucleotide barcodes are associated with a solid support. Each of the plurality of oligonucleotide barcodes associated with the same solid support may include the same sample label. Each of the plurality of sample labels of the plurality of oligonucleotide barcodes may include at least 6 nucleotides. Each of the plurality of oligonucleotide barcodes may include a cell label. Each of the plurality of cell labels of the plurality of oligonucleotide barcodes may include at least 6 nucleotides. The oligonucleotide barcodes associated with the same solid support may include the same cell label. The oligonucleotide barcodes associated with different solid supports may include different cell labels. Each of the plurality of extended barcoded nucleic acid molecules may include a cell label and a complement of the cell label. The complement of the cell label may include a reverse complement of the cell label or a 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, dimethylsulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salts, betaine, or any combination thereof. In some embodiments, the solid support may include synthetic particles. In some embodiments, the solid support may include a planar surface.

[0182] The sample may include a single cell, and the method may include associating a synthetic particle 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 particle with the single cell. Lysing the single cell may include heating the sample, contacting the sample with a detergent, altering the pH of the sample, or any combination thereof. In some embodiments, the synthetic particle and the single cell are in the same well. In some embodiments, the synthetic particle 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 disintegrable. 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, 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 the target binding region of the barcoded nucleic acid molecule itself 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 in 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 an extended barcoded nucleic acid molecule 620c. The extended barcoded nucleic acid molecule 620c may include a complement (e.g., reverse complement) of first molecular label 622rc, a complement (e.g., reverse complement) of cell label 624rc, and / or a 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, the intermolecular hybridization 600c1 and / or the extension 600c2 are performed in the presence of a high salt buffer and / or PEG. In some embodiments, the extension is performed using a DNA polymerase that lacks at least one of 5' to 3' and 3' to 5' exonuclease activities (e.g., Klenow fragment).

[0185] The single-stranded extended barcoded nucleic acid molecule 620cd may include barcodes (e.g., cell and molecular labels) at both the 5' and 3' ends of the target nucleic acid molecule (e.g., transcript), thereby allowing for more extensive analysis of the target nucleic acid molecule for sequence identification, transcript counting, alternative splicing analysis, mutation screening, and / or full-length sequencing, as 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 a single-labeled nucleic acid molecule 620c1 comprising first molecular label 622, cell label 624, first universal sequence 626 and partial cDNA 614c1 (whose length varies depending on the binding site of target-specific primer 648 in cDNA 614c). PCR1 600c4 may include amplifying the 3' region of the single-stranded, extended barcoded nucleic acid molecule 620cd comprising the universal oligonucleotide primer 646 and the target-specific primer 650, thereby generating a single-labeled nucleic acid molecule 620cas1 comprising the first molecular label 622, the cell label 624, the first universal sequence 626 and the partial antisense cDNA 614cas1 (whose length varies depending on the binding site of the target-specific primer 650 in the 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 a universal oligonucleotide primer 646 and a nested target-specific primer (e.g., target-specific primer 652 and / or target-specific primer 654). The target-specific primer 652 and / or target-specific primer 654 may include an overhang that may include or may be, for example, a second universal sequence 638 (e.g., a sequence of read 2, a universal PCR handle). PCR2 600c5 may include amplifying a single-stranded nucleic acid molecule 620c1 that includes the universal oligonucleotide primer 646 and the nested target-specific primer 654, thereby generating a single-labeled nucleic acid molecule 620c2 that includes the first molecular label 622, the cell label 624, the first universal sequence 626, the second universal sequence 638, and a partial cDNA 614c2 (whose length varies depending on the binding site of the nested target-specific primer 654 in the partial cDNA 614c1). PCR2 600c5 may include amplifying a single-labeled nucleic acid molecule 620cas1 that includes a universal oligonucleotide primer 646 and a nested target-specific primer 652, thereby generating a single-labeled nucleic acid molecule 620cas2 that includes 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 (whose length 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 regions, variable regions, diversity regions, 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 adaptors (e.g., P5 640 and P7 642) and sample indexes 644 (e.g., i5, i7) by 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 150bp x 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. PCR3 600c6 may 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 and / or B cells in a single cell platform may be investigated. In some embodiments, both 3' and 5' information of their transcripts may be captured in a single experiment. The methods disclosed herein may allow for V(D)J detection of both T and B cells (e.g., hypermutation). 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 of 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 of 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 include 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 a 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. The workflow may, in some embodiments, 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 the second molecular label, a complement (e.g., reverse complement) 624rc of the cell label, and / or a complement (e.g., reverse complement) 626rc of the 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 can include barcodes (e.g., cell and molecular labels) at both the 5' and 3' ends of the target nucleic acid molecule (e.g., transcript), thereby allowing for more extensive analysis of the target nucleic acid molecule for sequence identification, transcript counting, alternative splicing analysis, mutation screening, and / or full-length sequencing, as 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 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 a single-labeled nucleic acid molecule 620d1 comprising first molecular label 622, cell label 624, first universal sequence 626 and partial cDNA 614c1 (whose length varies depending on the binding site of target-specific primer 648 in cDNA 614c). PCR1 600d4 may include amplifying the 3' region of the single-stranded extended barcoded nucleic acid molecule 620dd, which comprises the universal oligonucleotide primer 646 and the target-specific primer 650, thereby generating a single-labeled nucleic acid molecule 620das1, which comprises the second molecular label 632, the cell label 624, the first universal sequence 626 and the partial antisense cDNA 614cas1 (whose length varies depending on the binding site of the target-specific primer 650 in the 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 a universal oligonucleotide primer 646 and a nested target-specific primer (e.g., target-specific primer 652 and / or target-specific primer 654). The target-specific primer 652 and / or target-specific primer 654 may include an overhang that may, for example, include a second universal sequence 638 (e.g., a sequence of read 2, a universal PCR handle). PCR2 600d5 may include amplifying a single-labeled nucleic acid molecule 620d1 that includes the universal oligonucleotide primer 646 and the nested target-specific primer 654, thereby generating a single-labeled nucleic acid molecule 620d2 that includes a first molecular label 622, a cell label 624, a first universal sequence 626, a second universal sequence 638, and a partial cDNA 614c2 (whose length varies depending on the binding site of the nested target-specific primer 654 in the cDNA 614c1). PCR2 600d5 may include amplifying a single-labeled nucleic acid molecule 620das1 that includes a universal oligonucleotide primer 646 and a nested target-specific primer 652, thereby generating a single-labeled nucleic acid molecule 620das2 that 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 (whose length 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 constant regions, variable regions, diversity regions, and / or junction regions 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 adaptors (e.g., P5 640 and P7 642) and sample indexes 644 (e.g., i5, i7) by 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 disclosure. Sequencing using 150bp x 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. PCR3 600d6 may 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 and / or B cells in a single cell platform may be investigated. In some embodiments, both 3' and 5' information of their transcripts may be captured in a single experiment. The methods disclosed herein may allow for V(D)J detection of both T 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 include 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 the 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 the 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 comprises extending the 3' end of the 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 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 include intermolecular hybridization of a single-stranded barcoded nucleic acid molecule 621 with a separate oligonucleotide barcode 634, as shown in the non-limiting exemplary schematic diagrams of Figures 6I-6J. The separate 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 a cDNA 614c. The workflow may include denaturing (600e3) extended barcoded nucleic acid molecule 620e1 to generate a 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, the intermolecular hybridization 600e1 and / or the extension 600e2 are performed in the presence of a high salt buffer and / or PEG. In some embodiments, the 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] The single-stranded extended barcoded nucleic acid molecule 620e1d and the single-stranded extended barcoded nucleic acid molecule 620e2d can include barcodes (e.g., cell and molecular labels) at both the 5' and 3' ends of the target nucleic acid molecule (e.g., transcript), thereby allowing for more extensive analysis of the target nucleic acid molecule for sequence identification, transcript counting, alternative splicing analysis, mutation screening, and / or full-length sequencing, as compared to analysis of a target nucleic acid molecule having only one barcode at one end. The single-stranded extended barcoded nucleic acid molecule 620e1d and the single-stranded extended barcoded nucleic acid molecule 620e2d can serve as templates for one or more amplification reactions (e.g., PCR). The 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 rounds of PCR amplification (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 round of amplification, such as, for example, the non-limiting exemplary amplification scheme shown in Figure 6K (PCR 600e4). PCR 600e4 may add sequencing adaptors (e.g., P5 640 and P7 642) and sample index 644 (e.g., i5, i7) by overhangs in primers 660, 662, and 664.PCR 600e4 may include amplifying single-stranded, extended barcoded nucleic acid molecule 620e1d, comprising primer 664 (annealing to the first universal sequence or its complement) and target-specific primer 660, thereby generating a single-labeled nucleic acid molecule 620e1c, comprising first molecular label 622, cell label 624, first universal sequence 626 and partial cDNA 614c1e (whose length varies depending on the binding site of target-specific primer 660 in cDNA 614c). PCR 600e4 may include amplifying a single-stranded extended barcoded nucleic acid molecule 620e2d that includes primer 664 (annealing to the first universal sequence or its complement) and target-specific primer 662, thereby generating a single-labeled nucleic acid molecule 620e2c that 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 614cas). Library amplicons 620e1c and / or 620e2c may be sequenced and subjected to downstream methods of the disclosure. Sequencing using 150bp×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 indexes 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 an immune receptor.

[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 and / or B cells in a single cell platform may be investigated. In some embodiments, both 3' and 5' information of the transcript may be captured in a single experiment. The methods disclosed herein may allow for V(D)J detection of both T and B cells (e.g., hypermutation). In some embodiments, both the 3' and 5' regions of the extended barcoded nucleic acid molecules 620e1d and / or 620e2d are amplified. In some embodiments, only the 5' regions of the extended barcoded nucleic acid molecules 620e1d and / or 620e2d are amplified. In some embodiments, only the 3' regions of the extended barcoded nucleic acid molecules 620e1d and / or 620e2d are 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 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 of 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 a 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 copies of the plurality of extended barcoded nucleic acid molecules comprises using a primer capable of hybridizing 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 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 an immune receptor, a variable region of an immune receptor, a diversity region of an immune receptor, a junction of a variable region and a diversity region of an immune receptor, or any combination thereof. The immune receptor may be a T cell receptor (TCR) and / or a B cell receptor (BCR) receptor. 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 the plurality of extended barcoded nucleic acid molecules, or products thereof. Obtaining sequence information may include attaching a sequencing adaptor to the plurality of extended barcoded nucleic acid molecules, or products thereof. Obtaining sequence information may include attaching a sequencing adaptor to the 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 the BCR heavy chain of the single cell. The sequence information of the BCR light chain and the BCR heavy chain may include the sequence of the complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the BCR light chain and / or the BCR heavy chain. The method may include pairing the BCR light chain and the 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 the 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%, 99%, 100%, 1 %, 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 the BCR heavy chain are paired according to the methods provided herein is at least or at most 0.000000001%, 0.00000001%, 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%, 102%, 103%, 1 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 may 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 the TCR beta chain of the single cell. In some embodiments, the sequence information of the TCR alpha chain and the TCR beta chain may include the sequence of the 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 may include 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 may include a plurality of single cells, and the method may include 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, the percentage of single cells of a sample in which the TCR alpha chain and the TCR beta chain are paired according to the methods provided herein is 0.000000001%, 0.00000001%, 0.0000001%, 0.00001%, 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 1 %, 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 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 the TCR beta chain are paired according to the methods provided herein is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 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%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 10 %, 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 TCR gamma chain and TCR delta chain of a single cell. The sequence information of TCR gamma chain and TCR delta chain may include the sequence of complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of TCR gamma chain and / or TCR delta chain. The method may include pairing TCR gamma chain and TCR delta chain of a single cell based on the obtained sequence information. The sample may include a plurality of single cells, and the method may include pairing TCR gamma chain and 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 the TCR gamma chain are paired according to the methods provided herein is 0.000000001%, 0.00000001%, 0.0000001%, 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%, 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%, 99%, 100%, 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 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%, 0.00001%, 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%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 10 %, 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] Kits for barcoding the 5' and 3' ends of nucleic acid targets The disclosure herein includes a kit. In some embodiments, the kit includes a plurality of oligonucleotide barcodes, each of the plurality of oligonucleotide barcodes including a molecular label and a target binding region, at least 10 of the plurality of oligonucleotide barcodes including different molecular label sequences; a reverse transcriptase; a template switching oligonucleotide including 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 includes a Klenow fragment. In some embodiments, the reverse transcriptase includes 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 switch oligonucleotide includes one or more 3' ribonucleotides, e.g., three 3' ribonucleotides. In some embodiments, the 3' ribonucleotide includes a guanine. In some embodiments, the kit comprises 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.

[0205] In some embodiments, the kit comprises a buffer. In some embodiments, the kit comprises a cartridge. In some embodiments, the kit comprises one or more reagents for a reverse transcription reaction. In some embodiments, the kit comprises 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. In some embodiments, the oligonucleotide barcodes comprise the same sample label and / or the same cell label. In some embodiments, each sample label and / or cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides. In some embodiments, each molecular label of the plurality of oligonucleotide barcodes comprises 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 disintegrable. 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, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.In some embodiments, the synthetic particle comprises a disintegrable 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. EXAMPLES

[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. Using the non-limiting exemplary V(D)J protocol described below, we have demonstrated 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 reaction mix. 5. Place the tubes 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 at 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 at 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, Calif.).

[0208] Self-hybridization 1. Shake the tubes at 1200 rpm for 5 minutes at 37C, 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 a 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 37C for 30 minutes at 1200 rpm. 5. Place the tube in 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 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 200 uL of PCR1 reaction mix. Pipette up and down gently 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 min, 15 cycles of (95°C for 30 sec, 60°C for 3 min, 72°C for 1 min), 72°C for 5 min. 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 pipette the PCR1 product into a new tube.

[0212] Cleanup of PCR1 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 2 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 still 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 2 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 the 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 4C or on ice if the next step is done on 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 min, 15 cycles of (95°C for 30 sec, 60°C for 3 min, 72°C for 1 min), 72°C for 5 min.

[0217] PCR2 Cleanup 1. For TCR and BCR products, add 30ul Ampure XP beads (0.6x the volume of PCR products) to the PCR1 product. For IR3' and 5', add 50ul 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 2 washes. 8. Allow the Ampure beads to air dry on the magnet with the lid open until no visible droplets are present. 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 done on the same day, or store at -20C until use. 12. Use the Qubit DNA HS Assay to measure the amount of eluted DNA and evaluate if dilution of the product is necessary for the next PCR. The PCR2 product must be diluted to 10ng / ul or less using elution buffer before proceeding to the final PCR to avoid overamplification.

[0218] Indexed PCR 1. In the Pre-PCR area, prepare the following reaction mix 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 min, 8 cycles of (98°C for 15 sec, 60°C for 30 sec, 72°C for 30 sec), 72C for 1 min.

[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 1.5ml 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 2 washes. 8. Allow the Ampure beads to air dry on the magnet with the lid open until no visible droplets are present. 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 done on 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 evaluate if dilution of the product is necessary for the next PCR. The PCR2 product must be diluted to 10ng / ul or less using elution buffer before proceeding to the final PCR to avoid overamplification.

[0220] Example 1 V(D)J Protocol This example demonstrates generating sequencing libraries for both the 3' and 5' ends of the mRNA targets of a targeted panel. In this example, sequencing libraries were generated for both the 3' and 5' ends of mRNA targets from a targeted 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, CDR3 rearrangement patterns were identified in lymphocytes from peripheral blood mononuclear cells of healthy donors.

[0221] Figures 8A-8C show non-limiting exemplary experimental results of capture and sequencing of 5' T cell receptor (TCR) V(D)J regions using the V(D)J protocol. The V(D)J hairpin protocol can include 3' and 5' amplifications performed on the same bead for captured single, resting peripheral blood mononuclear cell (PBMC) derived mRNA molecules. Figure 8A shows the annotation of cell types. 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 was comparable to other platforms such as Clonetech's scTCR profiling kit. Table 10 shows the TCR alpha / beta pairings achieved using this V(D)J protocol.

[0222] [Table 10]

[0223] Figures 9A-9B show non-limiting exemplary plots showing 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 in reverse transcription (RT). Hybridization time, buffer, and template switching (TS) oligo dT length were altered 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 respect 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 the annotation of cell types. 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, the 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 the TCR alpha / beta pairing and BCR heavy / 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 with 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 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 of ordinary skill 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 orders. Moreover, 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 diminishing the essential elements of the embodiments of the present disclosure. In connection with the use of substantially any plural and / or singular term herein, where appropriate in the context and / or application, those skilled in the art can convert from plural to singular and / or from singular to plural, and 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, and 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, it will be understood by those skilled in the art that where a specific number of introduced claim recitations are intended, such intent will be expressly set forth in the claim, and that in the absence of such recitation, no such intent 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 the 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" limits any particular claim that includes such introduced claim recitation 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.Moreover, even if a particular number of introduced claim recitations is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., an unmodified recitation without other modifiers such as "two recitations" 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, such a configuration is generally intended to mean as one of ordinary skill in the art would understand the convention (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, such construction is generally intended to be as one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or 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.). Additionally, it will be understood by those of ordinary skill 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 disclosure are described in terms of a Markush group, it will be recognized by those skilled in the art that the disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0229] As will be understood by those of skill in the art, for all purposes, e.g., with respect to the provision of the specification, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any recited range is readily recognizable as being fully descriptive and that the range can be divided into at least 2, 3, 4, 5, 10, etc. 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 of skill in the art, all expressions such as "up to," "at least," etc. refer to ranges that are inclusive of the recited numbers and that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those of skill in the art, ranges include each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 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. Yet another aspect of the present invention may be as follows. [1] A method for labeling a nucleic acid target 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 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; 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; and hybridizing the target binding region of extending 3' ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules, each of the plurality of extended barcoded nucleic acid molecules comprising a first molecular label and a second molecular label; A method comprising: [2] The method of [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] 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 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; 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; and hybridizing the target binding region of extending 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 a 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 a first molecular label or a second molecular label; The method according to any one of [2] to [3] above, wherein the step of determining the copy number of the nucleic acid target in the sample comprises a step of 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] The method according to [4], wherein the step of determining the copy number of the nucleic acid target in the sample comprises a step of 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; The method according to any one of [2] to [3] above, wherein the step of determining the copy number of the nucleic acid target in the sample comprises a step of 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 elongated 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 elongated barcoded nucleic acid molecules, or products thereof. [7] 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 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; 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; and hybridizing the target binding region of extending 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] The method according to [7], further 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] The method according to any one of [1] to [8] above, comprising a step of denaturing the plurality of barcoded nucleic acid molecules before 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) a target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules.

[10] The method according to any one of [4] to [9] above, comprising a step of denaturing the plurality of elongated barcoded nucleic acid molecules before amplifying the plurality of elongated barcoded nucleic acid molecules.

[11] The method according to any one of [7] to [9] above, wherein the step of determining the copy number of the nucleic acid target comprises a step of determining the copy number of each of the multiple 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 each sequence of the multiple nucleic acid targets.

[12] The method according to

[11] , wherein the step of determining the copy number of the nucleic acid target comprises a step of determining the copy number of each of the multiple 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 that include each sequence of the multiple nucleic acid targets.

[13] The method according to

[12] , wherein each sequence of the multiple nucleic acid targets comprises a partial sequence of each of the multiple nucleic acid targets.

[14] The method according to any one of [1] to

[13] above, wherein the sequence of the nucleic acid target in the multiple barcoded nucleic acid molecules comprises a partial sequence of the nucleic acid target.

[15] The method according to any one of [1] to

[14] above, 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] The method according to any one of [1] to

[15] above, wherein each of the extended barcoded nucleic acid molecules comprises a first molecular label, a second molecular label, a target binding region, and a complement of the target binding region.

[17] The method according to any one of [1] to

[16] above, wherein the complement of the target binding region is complementary to a portion of the target binding region.

[18] The method acco...

Claims

1. 1. A method for labeling a nucleic acid target 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 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, wherein the reverse transcriptase is capable of terminal transferase activity, and the template switch oligonucleotide comprises one or more 3' ribonucleotides, and the 3' ribonucleotide comprises a guanine; The complement of the target binding region of each barcoded nucleic acid molecule is (i) a target binding region of an oligonucleotide barcode among the plurality of oligonucleotide barcodes; (ii) the target binding region of the barcoded nucleic acid molecule itself, and / or (iii) target binding regions of different barcoded nucleic acid molecules among the plurality of barcoded nucleic acid molecules. and hybridizing the nucleic acid sequence with 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, the second molecular label being generated by extension of the 3' ends of the barcoded nucleic acid molecules in an extending step subsequent to the hybridizing step, and the sequence of the generated second molecular label being: (a) has a sequence that, when generated following hybridization of a complement of the target binding region of the barcoded nucleic acid molecule with (i) the target binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes, is distinct from the sequence of the first molecular label; (b) the complement of the target binding region of the barcoded nucleic acid molecule and (ii) the complement of the first molecular label when generated following hybridization of the barcoded nucleic acid molecule with its own target binding region; and / or (c) has a sequence that is distinct from the sequence of the first molecular label when generated following hybridization of the complement of the target binding region of the barcoded nucleic acid molecule with (iii) the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules. Steps and A method comprising:

2. 13. 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. (a) amplifying a 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 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; or (b) 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; 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; 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; or (c) amplifying the plurality of extended bar-coded nucleic acid molecules to generate a plurality of copies of the extended bar-coded nucleic acid molecules; 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 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; The method of claim 2.

4. 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 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, wherein the reverse transcriptase is capable of terminal transferase activity, and the template switch oligonucleotide comprises one or more 3' ribonucleotides, and the 3' ribonucleotide comprises a guanine; The complement of the target binding region of each barcoded nucleic acid molecule is (i) a target binding region of an oligonucleotide barcode among the plurality of oligonucleotide barcodes; (ii) the target binding region of the barcoded nucleic acid molecule itself, and / or (iii) target binding regions of different barcoded nucleic acid molecules among the plurality of barcoded nucleic acid molecules. and hybridizing the nucleic acid sequence with 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, the second molecular label being generated by extension of the 3' ends of the barcoded nucleic acid molecules in an extending step subsequent to the hybridizing step, and the sequence of the generated second molecular label being: (a) has a sequence that, when generated following hybridization of a complement of the target binding region of the barcoded nucleic acid molecule with (i) the target binding region of an oligonucleotide barcode of the plurality of oligonucleotide barcodes, is distinct from the sequence of the first molecular label; (b) the complement of the target binding region of the barcoded nucleic acid molecule and (ii) the complement of the first molecular label when generated following hybridization of the barcoded nucleic acid molecule with its own target binding region; and / or (c) has a sequence that is distinct from the sequence of the first molecular label when generated following hybridization of the complement of the target binding region of the barcoded nucleic acid molecule with (iii) the target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules. Steps and 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:

5. 5. The method of claim 4, 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.

6. 2. The method of claim 1, comprising denaturing the plurality of barcoded nucleic acid molecules prior to hybridizing a 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) a target binding region of a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules.

7. 4. The method of claim 3, comprising denaturing the plurality of extended barcoded nucleic acid molecules prior to amplifying the plurality of extended barcoded nucleic acid molecules.

8. (a) 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 the plurality of single-labeled nucleic acid molecules that include the respective sequences of the plurality of nucleic acid targets; (b) 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 the plurality of single-labeled nucleic acid molecules comprising the respective sequences of the plurality of nucleic acid targets, and 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 the plurality of single-labeled nucleic acid molecules comprising the respective sequences of the plurality of nucleic acid targets; (c) 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 the plurality of single-labeled nucleic acid molecules that comprise a respective sequence of the plurality of nucleic acid targets, wherein the sequence of each of the plurality of nucleic acid targets comprises a respective subsequence of the plurality of nucleic acid targets; or (d) 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 the plurality of single-labeled nucleic acid molecules comprising the respective sequences of the plurality of nucleic acid targets, 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 the plurality of single-labeled nucleic acid molecules comprising the respective sequences of the plurality of nucleic acid targets, wherein the sequence of each of the plurality of nucleic acid targets comprises a respective subsequence of the plurality of nucleic acid targets; The method according to claim 4.

9. (a) a sequence of the nucleic acid target in the plurality of barcoded nucleic acid molecules comprises a subsequence of the nucleic acid target; (b) the first molecular label is hybridized to a second molecular label after extending the 3' ends of the plurality of barcoded nucleic acid molecules; (c) each of the extended barcoded nucleic acid molecules comprises a first molecular label, a second molecular label, a target binding region, and a complement of the target binding region; (d) the complement of the target binding region is complementary to the target binding region; (e) the target binding region comprises a gene-specific sequence and / or a poly(dT) sequence; (f) hybridizing the complement of the target binding region of the barcoded nucleic acid molecule with the target binding region of the barcoded nucleic acid molecule itself 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; and / or (g) 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. (h) 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. (i) the template switch oligonucleotide comprises three 3′ ribonucleotides; (j) the reverse transcriptase comprises a viral reverse transcriptase; (k) the sample comprises a single cell; (l) the sample comprises a plurality of cells, a plurality of single cells, a tissue, a tumor sample, or any combination thereof; (m) each oligonucleotide barcode comprises a first universal sequence; and / or (n) the plurality of extended barcoded nucleic acid molecules comprises a first universal sequence and a complement of the first universal sequence; The method of claim 1.

10. (a) amplifying the plurality of extended barcoded nucleic acid molecules to generate a plurality of copies of the extended barcoded nucleic acid molecules comprises using a primer capable of hybridizing to a first universal sequence, or a complement thereof; or (b) 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 a complement thereof, and an amplification primer; (c) 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, wherein the amplification primer is a target-specific primer; (d) 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, wherein the amplification primer is a target-specific primer, and the target-specific primer specifically hybridizes to an immune receptor, a constant region of the immune receptor, a variable region of the immune receptor, a diversity region of the immune receptor, and / or a junction of the variable region and diversity region of the immune receptor; (e) 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 a complement thereof, and an amplification primer, wherein the amplification primer is a target-specific primer, and the target-specific primer specifically hybridizes 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 of the variable region and diversity region of an immune receptor, and the immune receptor is a T cell receptor (TCR) and / or a B cell receptor (BCR); and / or (f) 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, wherein the amplification primer is a target specific primer, wherein the target specific primer specifically hybridizes to an immune receptor, a constant region of the immune receptor, a variable region of the immune receptor, a diversity region of the immune receptor, and / or a junction of the variable region and diversity region of the immune receptor, wherein the immune receptor is a T cell receptor (TCR) and / or a B cell receptor (BCR), wherein the TCR comprises a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof, and wherein the BCR comprises a BCR heavy chain and / or a BCR light chain; The method according to claim 3.

11. (A) 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 that lacks at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity; and / or (B) the method comprises obtaining sequence information of a plurality of extended barcoded nucleic acid molecules, or products thereof; and / or (C) the method includes obtaining sequence information of a plurality of extended barcoded nucleic acid molecules, or products thereof; (a) obtaining sequence information by combining a sequencing adaptor with (i) a plurality of extended barcoded nucleic acid molecules, or products thereof; or (ii) a plurality of single-labeled nucleic acid molecules, or products thereof and / or (b) the step of obtaining sequence information comprises obtaining sequence information of a BCR light chain and a BCR heavy chain of the single cell; and / or (c) the step of obtaining sequence information comprises obtaining sequence information of the TCR alpha chain and the TCR beta chain of the single cell; and / or (d) obtaining sequence information includes obtaining sequence information of a TCR gamma chain and a TCR delta chain of the single cell; The method of claim 1.

12. (a) 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; (b) the complement of the molecular label comprises the reverse complement of the molecular label and / or the complement of the molecular label; (c) the plurality of barcoded nucleic acid molecules comprises barcoded deoxyribonucleic acid (DNA) molecules and / or barcoded ribonucleic acid (RNA) molecules; and / or (d) the nucleic acid target comprises a nucleic acid molecule; (e) at least ten of the plurality of oligonucleotide barcodes comprise different molecular label sequences; (f) each molecular label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; (g) a plurality of oligonucleotide barcodes are associated with the solid support; and / or (h) each of the plurality of oligonucleotide barcodes comprises a cell label; (i) 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, dimethylsulfoxide (DMSO), glycerol, formamide, 7-deaza-GTP, acetamide, tetramethylammonium chloride salts, betaine, or any combination thereof; (j) the solid support comprises a synthetic particle or a planar surface; and / or (k) the sample comprises a single cell, and the method comprises associating a synthetic particle comprising a plurality of oligonucleotide barcodes with the single cell in the sample; The method of claim 1.

13. (a) the method comprises lysing the single cells after associating the synthetic particles with the single cells, wherein lysing the single cells comprises heating the sample, contacting the sample with a detergent, altering the pH of the sample, or any combination thereof; and / or (b) Synthetic particles and single cells (i) in the same well, or (ii) in the same droplet; and / or (c) 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; (d) the synthetic particles are disintegratable; and / or (e) the synthetic particles include beads, the beads comprising: (i) 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; (ii) a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogels, paramagnetic materials, ceramics, plastics, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; or (iii) Collapsible Hydrogel Particles and / or (f) each of the plurality of oligonucleotide barcodes comprises a linker functional group, and the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other; The method of claim 12(k).

14. The viral reverse transcriptase is murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase; The single cell comprises an immune cell. the single cell comprises a B cell or a T cell; and / or The single cell comprises a circulating tumor cell.

10. The method of claim 9.

15. (a) the DNA polymerase comprises a Klenow fragment, and / or (b)(i) the sequence information of the BCR light chain and the BCR heavy chain includes the sequence of the complementarity determining region 1 (CDR1), CDR2, CDR3, or any combination thereof, of the BCR light chain and / or the BCR heavy chain; (ii) the method comprises pairing the BCR light chain and the BCR heavy chain of the single cell based on the obtained sequence information; and / or (iii) the sample comprises a plurality of single cells, and the method comprises pairing the BCR light chain and the BCR heavy chain of at least 50% of said single cells based on the obtained sequence information; and / or (c) (i) 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; (ii) the method comprises pairing the TCR alpha and beta chains of a single cell based on the sequence information obtained; and / or (iii) the sample comprises a plurality of single cells, and the method comprises pairing the TCR alpha and beta chains of at least 50% of said single cells based on the sequence information obtained; and / or (d)(i) the sequence information of the TCR gamma chain and the TCR delta chain includes 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; (ii) the method comprises pairing the TCR gamma chain and the TCR delta chain of the single cell based on the obtained sequence information; and / or (iii) 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. The method of claim 11.

16. (a) the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof; (b) the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof, wherein the mRNA encodes an immune receptor; (c) each of the multiple oligonucleotide barcodes associated with the same solid support comprises an identical sample label; (d) each of the plurality of oligonucleotide barcodes associated with the same solid support comprises an identical sample label, and each sample label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; (e) each cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; (f) oligonucleotide barcodes associated with the same solid support contain the same or different cell markers; (g) each of the plurality of extended barcoded nucleic acid molecules comprises a cell marker and a complement of the cell marker; and / or (h) each of the plurality of extended barcoded nucleic acid molecules comprises a cell marker and a complement of the cell marker, wherein the complement of the cell marker comprises a reverse complement of the cell marker and / or a complement of the cell marker; The method of claim 12.

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