RU2790291C2 - High throughput sequencing of polynucleotide libraries and transcriptome analysis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for obtaining a polynucleotide library is presented. A polynucleotide library containing a plurality of barcoded polynucleotides obtained by this method is also presented. The library used to sequence one or more of a variety of target polynucleotides. In addition, the invention relates to a method for sequencing and methods for analyzing a transcriptome.

EFFECT: performing transcriptomic sequencing, such as RNA sequencing, obtaining a picture of the transcriptome of individual cells in combination with full-length immune receptor sequences so that information about the immune repertoire and the transcriptome of the cell can be obtained.

137 cl, 5 dwg, 5 tab, 8 ex

Description

Cross-reference to related applications

[0001] This application claims priority under U.S. Provisional Application No. 62/511,949, filed May 26, 2017, entitled "HIGH CAPACITY POLYNUCLEOTIDE LIBRARY SEQUENCING AND TRANSCRIPTOME ANALYSIS", the contents of which are incorporated herein by reference in their entirety. .

Inclusion in the description of the invention of information by reference to the sequence listing

[0002] This application is filed with the sequence listing in electronic format. The sequence list is provided as a text file 735042011740SeqList.txt, created on May 25, 2018, which is 444416 bytes in size. The Sequence Listing information in electronic format is incorporated herein by reference in its entirety.

The field of technology to which the invention belongs

[0003] The present disclosure relates to methods for sequencing target genes and single cell barcoding in combination with single cell gene expression analysis. In some embodiments, the target gene is an immune molecule, such as an antibody or TCR. In some embodiments, the methods can be used to perform transcriptome sequencing, such as RNA sequencing, to represent the individual cell transcriptome in conjunction with full-length immune receptor sequences such that information about the cell's immune repertoire and transcriptome can be obtained. The present invention also relates to polynucleotide libraries that can be used for transcriptome and immune system molecule analysis, such as antibodies or TCR, and for sequencing.

State of the art

[0004] Determining the transcriptome content of a cell or tissue (i.e., "gene expression profiling") provides a method for functional analysis of normal and diseased cells or tissues and includes providing information characterizing the "state" of the cell or tissue or identifying characteristics of cell subpopulations. Existing single cell transcriptome sequencing techniques are limited in their throughput and/or unable to capture full-length immune receptor sequences. Thus, improved methods are needed. Methods and compositions are provided that meet such needs.

The essence of the invention

[0005] Methods are provided for obtaining a polynucleotide library from single cells or multiple cells, which not only provide one or more full-length barcoded target sequences or their individual fragments, but also jointly generate a plurality of barcoded polynucleotide sequences, the totality of which, in to a large extent, is the transcriptome or genome of a cell or a plurality of cells. Barcoding makes it possible to analyze and quantify the expression or determine the presence of polynucleotides derived from the same cell. Also provided are polynucleotide libraries produced by the methods of the present invention. Also provided are methods for analyzing the transcriptome of a single cell or multiple cells, and combining transcriptome analysis with analysis of the target sequence(s).

[0006] In some embodiments, the methods include obtaining a polynucleotide library that includes adding a second adapter to each of a plurality of barcoded single stranded polynucleotides at or near the end opposite the first adapter attached to each of the single stranded barcoded polynucleotides, wherein the plurality single-stranded barcoded polynucleotides includes: (i) one or more single-stranded target polynucleotides, including an amplicon of one or more target polynucleotides, or complementary to those present in a single cell belonging to a population of cells; and (ii) a collection of single-stranded polynucleotides, each of which contains an amplicon of the polynucleotide or complementary to that, in the cell; and where each of the plurality of barcoded single stranded polynucleotides includes a carrier barcode that is the same for all polynucleotides (i) and (ii) derived from the same cell belonging to the cell population.

[0007] In some embodiments, the plurality of barcoded single-stranded polynucleotides includes polynucleotides (i) and (ii) from a plurality of cells from a population of cells. In some embodiments, the plurality of barcoded single strand polynucleotides further comprises a molecular barcode that is unique to each single stranded polynucleotide or amplified product thereof.

[0008] In some embodiments, the collection of single-stranded polynucleotides from each cell of the cell population collectively comprises complementary DNA (cDNA) strands of a transcriptome or partial transcriptome. In some embodiments, the transcriptome or partial transcriptome collectively comprise at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the transcripts present in the cell genome.

[0009] In some embodiments of the method, each of the barcoded single-stranded polynucleotides has a size that is greater than or greater than about 50 base pairs, greater than 100 base pairs, or greater than 200 base pairs. In some embodiments, each of the barcoded single stranded polynucleotides is from or about 50 base pairs (bp) to 1500 bp, from 50 bp to about 50 bp. up to 1250 b.p., from 50 b.p. up to 1000 b.p., from 50 b.p. up to 750 b.p., from 50 b.p. up to 500 b.p., from 100 b.p. up to 1500 b.p., from 100 b.p. up to 1250 b.p., from 100 b.p. up to 1000 b.p., from 100 b.p. up to 750 b.p. from 100 n.p. up to 500 b.p., from 200 b.p. up to 1500 n.p., from 200 n.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p., or from 250 b.p. up to 500 b.p.

[0010] In some embodiments of the method, the second adapter is added to a homogeneous mixture containing a plurality of single-stranded barcoded polynucleotides.

[0011] In some embodiments, the implementation of the first adapter contains a bar code of the media.

[0012] In some embodiments of the methods for obtaining a polynucleotide library, the method includes: (a) lysing cells in each of a plurality of media, where each of these media contains a cell from a sample containing a population of cells; (b) producing in each carrier a plurality of complementary polynucleotides, said production of said plurality of complementary polynucleotides comprising (i) producing one or more target polynucleotides complementary to one or more target polynucleotides present in the cell using one or more specific for target primers; and (ii) obtaining a collection of polynucleotides, each of which is complementary to a polynucleotide present in the cell. In some embodiments, the collection of polynucleotides (ii) is made using random and/or degenerate and/or non-specific oligoprimers. In some embodiments, the collection of polynucleotides (ii) is made using oligo-dT primers.

[0013] In some embodiments, each of said carriers further comprises a plurality of oligonucleotides with a molecular barcode, a pool of oligonucleotides with a carrier barcode, and optionally a first adapter, the method further comprising: (c) attaching to a plurality of optionally to each of a plurality of complementary polynucleotides, one of a plurality of oligonucleotides with a molecular barcode, resulting in a plurality of barcoded polynucleotides, such as polynucleotides with a molecular barcode, each of which contains a molecular barcode, where, optionally, a specific the molecular barcode is different from the molecular barcodes contained in other barcoded polynucleotides within the set and/or is a unique molecular barcode; (d) attaching a single or one of a pool of carrier-barcoded oligonucleotides or amplified products thereof to a plurality, optionally each, of the barcoded polynucleotides, thereby forming a plurality of double-barcoded polynucleotides, wherein each of the polynucleotides with double barcodes belonging to the same media contain the same media barcode. In some embodiments, step (d) comprises attaching a single or one of a pool of carrier-barcoded oligonucleotides, or an amplified product thereof, and a first adapter, or one of a pool of first adapters, or an amplified product thereof, to a plurality, optionally each, of the polynucleotides with molecular barcode, thereby generating a plurality of double-barcoded polynucleotides, optionally single-stranded double-barcoded polynucleotides, each containing a molecular barcode and a carrier barcode, each of the double-barcoded polynucleotides belonging to the same media contains the same barcode.

[0014] In some embodiments, the methods further comprise (e) generating a single-stranded amplicon for a plurality of double-barcoded polynucleotides, optionally for each of the plurality, and/or (f) adding a second adapter to each of the single-stranded amplicons, which means adding adapter to a double-barcoded single-stranded polynucleotide, wherein the first adapter and the second adapter are present at or near opposite ends of each of the double-barcoded single-stranded polynucleotides.

[0015] In some embodiments, methods for obtaining a library of polynucleotides include the following steps: (a) lysing cells in each of a plurality of carriers, where each of said carriers contains a cell from a sample containing a population of cells, a plurality of oligonucleotides with a molecular barcode, and a single oligonucleotide or a pool of oligonucleotides with a carrier barcode and optionally a first adapter or a pool of first adapters; (b) producing in each carrier a plurality of complementary polynucleotides, said producing said plurality including, but not limited to (i) producing one or more target polynucleotides complementary to one or more target polynucleotides present in the cell; and (ii) obtaining a collection of polynucleotides, each of which is individually complementary to a polynucleotide in the cell; (c) attaching to each complementary polynucleotide one of a plurality of oligonucleotides with a molecular barcode, resulting in a plurality of polynucleotides with a barcode, each of which contains a unique molecular barcode; (d) attaching a single or one of the pool of barcoded oligonucleotides of the carrier or amplified product thereof and a first adapter or one of the pool of first adapters or amplified product thereof to each of the barcoded polynucleotides, resulting in a plurality of double-barcoded polynucleotides -code, optionally single-stranded double-barcoded polynucleotides, wherein each of the double-barcoded polynucleotides contains a molecular barcode and a carrier barcode, and each of the double-barcoded polynucleotides belonging to the same carrier contains the same media barcode; (e) obtaining a single-stranded amplicon for each of the plurality of double-barcoded polynucleotides; and (f) adding a second adapter to each of the single-stranded amplicons, which means adding a second adapter to the double-barcoded single-stranded polynucleotide, wherein the first adapter and the second adapter are located at or near opposite ends of each of the double-barcoded single-stranded polynucleotides. code.

[0016] In some embodiments, the adapter, such as the first adapter, contains an oligonucleotide with a carrier barcode. In some embodiments, the carrier-barcoded oligonucleotide comprises a first adapter. In some embodiments, the collection of polynucleotides from each cell of the cell population collectively contains sequences that are complementary to transcriptome or partial transcriptome transcripts of the cell. In some embodiments, the transcriptome or partial transcriptome contains at least 60%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99%, or 100% of the transcripts present in cell genome.

[0017] In some embodiments, one or more of the target polynucleotide(s) and/or the polynucleotide in the cell is DNA. In some embodiments, one or more of the target polynucleotide(s) of (i) and/or the polynucleotide of (ii) from which the amplicon in (i) and/or the amplicon in (ii) is derived, is(are) DNA. In some embodiments, one or more of the target polynucleotide(s) and/or the polynucleotide present in the cell is RNA, such as mRNA. In some embodiments, one or more of the target polynucleotide(s) of (i) and/or the polynucleotide of (ii) from which the amplicon in (i) and/or the amplicon in (ii) is derived, is(are) RNA, such as mRNA.

[0018] In some embodiments, each or one or more complementary polynucleotides (b) is a cDNA. In some embodiments, each or one or more of the barcoded single stranded polynucleotides is a cDNA strand.

[0019] In some embodiments of the methods, the first adapter and/or the second adapter comprise at least one universal priming site (also: primer site). In some embodiments, the first adapter and the second adapter are different; and/or the first adapter contains a first universal priming site and the second adapter contains a second universal priming site, and where, optionally, the first universal priming site and the second universal priming site are different. In some embodiments, the first universal priming site and/or the second universal priming site is or contains a P7(C7) priming site or a continuous portion thereof, or a P5 priming site or a continuous portion thereof, and where, optionally, the continuous portion is sufficient for annealing complementary sequence. In some embodiments, the first universal priming site is or contains a P7 (C7) priming site or a continuous portion thereof, and the second universal priming site is or contains a P5 (C5) priming site or a continuous portion thereof. In some embodiments, the P7 priming site (C7) contains the sequence AGATCGGAAGAGCACACGTCTGAACTCCA (SEQ ID NO: 77) or is a contiguous portion thereof. In some embodiments, the P5 priming site contains the sequence AGATCGGAAGAGCGTCGTGTAGGGAAAAGAGTGTAGATCTCGGTGGTCGCC GTATCATT (SEQ ID NO: 78) or is a contiguous portion thereof. In some embodiments, the implementation of the contiguous part contains at least or at least about 15, 20, 25 or 30 nucleotides in length. In some embodiments, the P5 priming site is a contiguous fragment as indicated in SEQ ID NO: 25 (AGATCGGAAGAGCGTCGTGT).

[0020] In some embodiments, adding a second adapter comprises hybridizing the splint oligonucleotide to each of the barcoded single stranded polynucleotides in the presence of an oligonucleotide comprising a second universal priming site, wherein the splint oligonucleotide contains (i) a sequence complementary to the second universal a priming site; and (ii) a degenerate overhang sequence capable of randomly annealing the 3' end of the barcoded single stranded polynucleotide. In some cases, prior to hybridization, the splint oligonucleotide and the oligonucleotide containing the second universal priming site are annealed to form an adapter duplex with the splint polynucleotide. In some aspects, the degenerate overhang sequence contains the sequence (N) _3-12 where N is any nucleotide.

[0021] In some of any such embodiments, the degenerate overhang sequence comprises the sequence NNNNNN, where N is any nucleotide (SEQ ID NO: 24). In some embodiments, the implementation hinge (splint) oligonucleotide contains the sequence ACACGACGCTCTTCCGATCTNNNNNN, where N is any amino acid (SEQ ID NO: 26). In some of any such embodiments, the oligonucleotide containing the second universal priming site contains the sequence AGATCGGAAGAGCGTCGTGT (SEQ ID NO: 25).

[0022] In some of any such embodiments, the carrier barcode oligonucleotide contains at least or at least about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 nucleotides. In some of any such embodiments, the carrier-barcoded oligonucleotide contains from or about 10 to 30 nucleotides. In some of any such embodiments, the oligonucleotide containing the carrier barcode contains a degenerate sequence. In some of any such embodiments, the carrier barcode-containing oligonucleotide comprises the sequence (N) _14-17 where N is any nucleotide, and where, optionally, at least one or two Ns in the sequence are W, where W is adenine or thymine. In some of any such embodiments, the carrier barcode oligonucleotide contains the sequence NNNNWNNNNWNNNN (SEQ ID NO: 80), WNNNNWNNNNWNNNN (SEQ ID NO: 81), NWNNNWNNNNWNNNN (SEQ ID NO: 82), or NNWNNNNWNNNNWNNNN (SEQ ID NO: 83), where N is any nucleotide and W is adenine or thymine.

[0023] In some of any such embodiments, each carrier comprises a pool of first adapters, wherein each carrier-barcoded oligonucleotide from the pool of first adapters contains at least one offset (shift) base or additional base relative to at least one other oligonucleotide with a carrier barcode in the pool. In some of any such embodiments, the carrier barcode oligonucleotides from the first adapter pool comprise the sequences NNNNWNNNNWNNNN (SEQ ID NO: 80), WNNNNWNNNNWNNNN (SEQ ID NO: 81), NWNNNWNNNNWNNNN (SEQ ID NO: 82) and NNWNNNNWNNNNWNNNN ( SEQ ID NO: 83), where N is any nucleotide and W is adenine or thymine.

[0024] In some of any such embodiments, in step (d), the method further comprises amplifying a single carrier barcoded oligonucleotide or pool of oligonucleotides or a single adapter or pool of first adapters, wherein the first adapters are a single barcoded oligonucleotide or pool of oligonucleotides. carrier, and the amplification is carried out before or simultaneously with the addition of the oligonucleotide with a carrier barcode to the polynucleotide with a molecular barcode. In some embodiments, attachment of a carrier barcode oligonucleotide comprises hybridizing a region of the carrier barcode oligonucleotide with a region of each of the complementary polynucleotides or with a region of each of the molecular barcode polynucleotides containing the molecular barcode. In some cases, the region contains a 3' labeled/labeled polynucleotide complementary to the 3' end region of the molecular barcode of the barcoded polynucleotides. In some embodiments, the region contains a 3' labeled polynucleotide complementary to the 5' end region of the molecular barcoded oligonucleotide.

[0025] In some of any such embodiments, in step (b), one or more target polynucleotide(s) is obtained by reverse transcription of the target(s) polynucleotide(s) in the presence of reverse transcriptase and one or more target polynucleotide(s). specific primers complementary to the target sequence of the target polynucleotide(s); and/or a collection of polynucleotides is obtained by reverse transcription of polynucleotides, such as polynucleotide transcripts, in a cell in the presence of reverse transcriptase and one or more transcriptome primers complementary to a polynucleotide, such as a polynucleotide transcript, in a cell.

[0026] In some of any such embodiments, the one or more target polynucleotide(s) comprises a polynucleotide of an immune system molecule or chain thereof. In some of any such embodiments, the one or more target polynucleotide(s) comprises at least two target polynucleotides, each of which comprises an immune molecule chain polynucleotide.

[0027] In some of any such embodiments, the one or more target polynucleotide(s) comprises a TCR polynucleotide or chain thereof. In some of any such embodiments, the one or more target polynucleotides comprise a first T cell receptor alpha (TCRα) polynucleotide and a second T cell receptor (TCRβ) polynucleotide. In some embodiments, the one or more target polynucleotide(s) comprises a first polynucleotide belonging to the T cell receptor gamma (TCRγ) and a second polynucleotide belonging to the T cell receptor delta (TCRdelta).

[0028] In some of any such embodiments, the one or more target polynucleotide(s) comprises an antibody polynucleotide or chains thereof. In some embodiments, the one or more target polynucleotide(s) comprises a first polynucleotide belonging to an immunoglobulin heavy chain (IgH) polynucleotide and a second polynucleotide belonging to an immunoglobulin light chain (IgL) polynucleotide. In some embodiments, one or more target-specific primers and/or one or more transcriptome primers contain a poly(T) sequence.

[0029] In some of any such embodiments, the one or more transcriptome primers comprise a mixture of random hexamer oligonucleotide primers. In some embodiments, the one or more target-specific primers include one or more primers that are complementary to the sequence(s) of the target sequence(s) of the target polynucleotide. In some cases, one or more target-specific primers contain at least two primers, such as a first primer and a second primer. In some embodiments, the one or more target-specific primers comprise primers for the target sequence from a plurality of target polynucleotides, each encoding an immune molecule or chain thereof. In some aspects, the immune system molecule is a T cell receptor or an antibody.

[0030] In some of any such embodiments, at least the first primer is complementary to the target polynucleotide sequence of the first strand of the immune molecule, and the second primer is complementary to the target sequence of the polynucleotide of the second strand of the immune molecule. In some embodiments, the first and second primers are complementary to the target sequences of different polynucleotides of the TCR chain of the TCR molecule.

[0031] In some of any such embodiments, the first primer is complementary to the target sequence of the TCRalpha polynucleotide sequence and the second primer is complementary to the target sequence of the TCRbeta polynucleotide sequence; or the first primer is complementary to the target sequence of the TCRgamma polynucleotide sequence and the second primer is complementary to the target sequence of the TCRdelta polynucleotide sequence. In some aspects, the target polynucleotide sequence of the TCR chain is a constant region sequence.

[0032] In some of any such embodiments, the first primer is complementary to the target sequence of the TCRalpha polynucleotide constant region sequence and the second primer is complementary to the TCRbeta polynucleotide constant region sequence; or the first primer is complementary to the target sequence of the TCRgamma polynucleotide constant region sequence and the second primer is complementary to the target sequence of the TCRdelta polynucleotide constant region sequence. In some embodiments, at least the first and second primers are complementary to target polynucleotide sequences of different antibody chains. In some embodiments, the first primer is complementary to the target immunoglobulin heavy chain (IgH) polynucleotide sequence and the second primer is complementary to the target immunoglobulin light chain (IgL) polynucleotide sequence. In some embodiments, the target polynucleotide sequence of the antibody chain is a constant region sequence.

[0033] In some of any such embodiments, the first primer is complementary to the target heavy chain constant region (CH) polynucleotide sequence and the second primer is complementary to the target light chain constant region (CL) polynucleotide sequence. In some instances, the CH polynucleotide target sequence is IgM, IgD, IgA, IgE, or IgG, or combinations thereof; and/or the target sequence of the CL polynucleotide sequence is from Igkappa, Iglambda, or combinations thereof.

[0034] In some of any such embodiments, the one or more target polynucleotide(s) comprises a full-length coding sequence. In some of any such embodiments, one or more target polynucleotide(s) and a collection of polynucleotides are produced in the vehicle in the same reaction volume.

[0035] In some of any such method embodiments in step (b), generating a plurality of complementary polynucleotides involves the use of a non-template terminal transferase, where three or more non-template nucleotides, ribonucleotides, or analogues thereof are added to the 3' end of each complementary polynucleotide produced. In some cases, the non-template terminal transferase is a reverse transcriptase or a polymerase. In some aspects, the non-template terminal transferase is a reverse transcriptase, where the reverse transcriptase is selected from the list: Superscript II reverse transcriptase, Maxima reverse transcriptase, Protoscript II reverse transcriptase, Maloney murine leukemia virus (MMLV-RT) reverse transcriptase, HighScriber reverse transcriptase, reverse transcriptase avian myeloblastosis virus (AMV), any reverse transcriptase including terminal deoxynucleotidyl transferase activity, or combinations thereof.

[0036] In some of any such method embodiments in step (c), the attachment comprises hybridizing a region of one of the plurality of molecular barcode oligonucleotides to three or more non-template nucleotides of each of the complementary polynucleotides. In some embodiments, the plurality of molecular barcode oligonucleotides are provided as a plurality of template-switched oligonucleotides, each including a 3' portion complementary to three or more non-template nucleotides. In some cases, the template switch oligonucleotide further includes a 5'-terminal region complementary to the 3'-labeled polynucleotide of the first adapter carrying the carrier barcode. In some cases, the template switch oligonucleotide also contains a 5'-terminal region complementary to the first adapter fragment, the first adapter containing the carrier barcoded oligonucleotide.

[0037] In some of any such embodiments, the reverse transcriptase has template switching activity; at least some of the many complementary polynucleotide chains produced contain a 3' overhang containing three or more non-template nucleotides; the plurality of molecular barcode oligonucleotides are provided as a plurality of template-switched oligonucleotides, each containing (1) a 5' end region that is complementary to a 3'-labeled oligonucleotide comprising a first adapter and a carrier barcode oligonucleotide, (2 ) a molecular barcode and (3) a 3' fragment complementary to three or more non-template nucleotides of the 3'overhang; wherein the template switch oligonucleotide serves as a template for reverse transcriptase such that a molecular barcode is included in each complementary polynucleotide to produce molecular barcoded polynucleotides.

[0038] In some of any such embodiments, a 3' fragment complementary to three or more non-template nucleotides includes a nucleotide, a ribonucleotide, or an analog thereof. In some of any such embodiments, the three or more non-template nucleotides include three or more C-nucleotides, and the 3' fragment complementary to the three or more non-template nucleotides contains one or more G-nucleotides, a ribonucleotide, or an analog thereof.

[0039] In some of any such embodiments, the template switch oligonucleotide also contains a 3'-modified nucleotide that blocks extension of the template switch oligonucleotide by reverse transcriptase or DNA polymerase. In some cases, the modification is a deoxy, phosphate, amino, or alkyl modification of the 3'-terminal nucleotide.

[0040] In some of any such method embodiments, step (d) further comprises extending each of the plurality of complementary polynucleotides after addition. In some embodiments, step (d) further comprises extending each of the plurality of double-barcoded molecular polynucleotides after addition to produce a plurality of double-barcoded polynucleotides.

[0041] In some of any such embodiments, the carrier is a well, emulsion, drop, or microcapsule.

[0042] In some of any such embodiments, prior to step (e), the method comprises combining the contents of two or more of the plurality of carriers to form a homogeneous mixture comprising two or more of the plurality of double-barcoded single-stranded polynucleotides. In some aspects, combining the contents of a plurality of carriers includes breaking two or more carriers from the plurality and combining single-stranded double-barcoded polynucleotides from the two or more carriers.

[0043] In some embodiments, prior to step (e), the method includes selecting or purifying single-stranded double-barbed polynucleotides having a size greater than or greater than about 50 base pairs, greater than 100 base pairs, or 200 base pairs. In some embodiments, the method comprises prior to step (e) selecting or purifying double-barcoded single-stranded polynucleotides having a size of from or about 50 base pairs (bp) to 1500 bp, from 50 bp to 1500 bp. up to 1250 b.p., 50 b.p. up to 1000 b.p., from 50 b.p. up to 750 b.p., from 50 b.p. up to 500 b.p., from 100 b.p. up to 1500 b.p., from 100 b.p. up to 1250 b.p., from 100 b.p. up to 1000 b.p., from 100 b.p. up to 750 b.p., from 100 b.p. up to 500 b.p., from 200 b.p. up to 1500 b.p. from 200 n.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p. or from 250 n.p. up to 500 b.p.

[0044] In some of any such embodiments, the double-barcoded single-stranded polynucleotides comprise, in the order (5' to 3'): first adapter, carrier barcode, molecular barcode, and second adapter. In some of any such embodiments, the first adapter is located at or near the 5' region of the single-stranded barcoded polynucleotide, optionally the double-barcoded single-stranded polynucleotide. In some of any such embodiments, the second adapter is located at or near the 3' region of the single-stranded barcoded polynucleotide, optionally the double-barcoded single-stranded polynucleotide.

[0045] In some of any such method embodiments, one or more of steps (a) to (f) is carried out in solution and/or in the absence/presence of a solid support, where optionally the support is a bead. In some of any such embodiments, at least steps (c) and (d) are carried out in solution and/or in the absence/presence of a solid support, where optionally the support is a bead. In some embodiments, each of steps (a)-(e) is carried out in solution and/or in the absence/presence of a solid support, where optionally the support is a bead.

[0046] In some of any such embodiments, the cell population comprises at least or at least about 1×10 ³ , 5×10 ³ , 1×10 ⁴ , 5×10 ⁴ , 1×10 ⁵ , 5×10 ⁵ , 1×10 ⁶ or 5×10 ⁶ cells. In some embodiments, the cell population is derived from a biological sample obtained from a subject. In some examples, the sample biological material is or contains whole blood, buffy coat, peripheral blood mononuclear cells (PBMC), unfractionated T cells, lymphocytes, leukocytes, an apheresis product, or a leukapheresis product.

[0047] In some of any such embodiments, the cell population comprises immune cells. In some embodiments, immune cells include lymphocytes or antigen presenting cells. In some embodiments, the immune cell is a lymphocyte or subtype thereof, a B cell or subtype thereof, a T cell or subtype thereof, or a combination thereof. In some examples, the immune system cell is a T cell, which is a CD4+ and/or CD8+ T cell.

[0048] In some of any such embodiments, the cell population is enriched in or contains central memory T cells, effector memory T cells, naive T cells, stem central memory T cells, effector T cells, and regulatory T cells. In some of any such embodiments, the cell population is enriched in memory B cells, naive B cells, or plasmablast B cells.

[0049] In some of any such embodiments, the subject is a human. In some embodiments, the subject has cancer, an infection, or an autoimmune condition. In some cases, the infection is a viral, bacterial or fungal infection.

[0050] In some of any such embodiments, the method further comprises amplifying a plurality of barcoded single strand polynucleotides, thereby generating a plurality of polynucleotide templates. In some of any such embodiments, amplification of a plurality of single-stranded barcode polynucleotides is performed in the presence of a first primer set including a first primer complementary to a first adapter sequence and a second primer complementary to a second adapter sequence. In some cases, the first and/or second primer is a universal primer. In some cases, the first and/or second primer is complementary to the P7 (C7) priming site or a continuous fragment thereof, or to the P5 (C5) priming site or a continuous fragment thereof. In some cases, the first primer is complementary to the P7 priming site (C7) or a continuous fragment thereof, and the second primer is complementary to the P5 priming site (C5) or a continuous fragment thereof. In some embodiments, the primer complementary to the P7 priming site (C7) or a contiguous fragment thereof has or contains the sequence CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 39); and/or a primer complementary to the P5(C5) priming site, or a continuous fragment thereof, contains the sequence ACACGACGCTCTTCCGATCT (SEQ ID NO: 27). In some of any such embodiments, the first and/or second primer further comprises a sequencing adapter. In some embodiments, the primer complementary to the P7(C7) priming site or a continuous fragment thereof further comprises the sequence CAAGCAGAAGACGGCATACGAGAT [NNNNNN] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 28); and/or the primer complementary to the P5(C5) priming site, or a continuous fragment thereof, contains the sequence AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACCTCTCTTCC (SEQ ID NO: 76).

[0051] In some embodiments, the method further comprises purifying each of the plurality of single-stranded barcoded polynucleotides, optionally double-barcoded single-stranded polynucleotides.

[0052] A polynucleotide library is provided containing a plurality of barcoded polynucleotides produced by the method of any of the described embodiments. Also provided is a polynucleotide library comprising a plurality of barcoded polynucleotides, wherein the plurality of barcoded polynucleotides comprises (i) one or more target polynucleotides comprising an amplicon of one or more target polynucleotides present in a cell taken from a population of cells; and (ii) a collection of polynucleotides, each containing an amplicon of a cellular polynucleotide, where each barcoded polynucleotide contains a first adapter including a first universal priming site complementary to the first universal primer; a carrier-barcoded oligonucleotide comprising a carrier barcode, wherein the carrier barcode is the same for all barcoded polynucleotides (i) and (ii) belonging to the same cell taken from a population of cells; and a second adapter sequence comprising a second universal priming site complementary to the second universal primer.

[0053] In some embodiments, each of the plurality of barcoded polynucleotides contains a molecular barcode that is unique to each polynucleotide or polynucleotide template. In some aspects, the collection of barcoded polynucleotide templates from each cell of the cell population collectively comprises or complementary DNA strands (cDNA) of the transcriptome or partial transcriptome. In some embodiments, the transcriptome or partial transcriptome collectively comprise at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the transcripts present in the cell genome.

[0054] In some of any such embodiments, each of the barcoded polynucleotides has a size that is greater than or approximately greater than 50 base pairs, greater than 100 base pairs, or greater than 200 base pairs. In some embodiments, each of the barcoded single stranded polynucleotides is from or about 50 base pairs (bp) to 1500 bp, from 50 bp to about 50 bp. up to 1250 b.p., from 50 b.p. up to 1000 b.p., from 50 b.p. up to 750 b.p., from 50 b.p. up to 500 b.p., 100 b.p. up to 1500 b.p., 100 b.p. up to 1250 b.p., 100 b.p. up to 1000 b.p., 100 b.p. 750 n.p., 100 n.p. up to 500 b.p., from 200 b.p. up to 1500 b.p., from 200 b.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p. or from 250 n.p. up to 500 b.p.

[0055] In some of any such embodiments, the first adapter contains a carrier barcode. In some embodiments, barcoded polynucleotides are single stranded. In some embodiments, barcoded polynucleotides are double stranded. In some embodiments, the first adapter and the second adapter are different.

[0056] In some of any such embodiments, the first universal priming site and/or the second universal priming site is or comprises a P7(C7) priming site or a continuous portion thereof, or a P5(C5) priming site or a continuous portion thereof, wherein, optionally, a continuous portion of these is sufficient to anneal the complementary sequence. In some embodiments, the first universal priming site is or contains a P7(C7) priming site or a continuous portion thereof, and the second universal priming site is or contains a P5(C5) priming site or a continuous portion thereof. In some aspects, the P7(C7) priming site contains or is a contiguous portion of the sequence AGATCGGAAGAGCACACGTCTGAACTCCA (SEQ ID NO: 77). In some examples, the P5 priming site contains the sequence AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT (SEQ ID NO: 78) or is a contiguous portion thereof.

[0057] In some of any such embodiments, the contiguous portion is at least or at least about 15, 20, 25, or 30 nucleotides in length. In some embodiments, the P5 priming site is a contiguous portion as indicated in SEQ ID NO: 25 (AGATCGGAAGAGCGTCGTGT).

[0058] In some of any such embodiments, the oligonucleotide containing the carrier barcode contains at least or at least about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 nucleotides. In some embodiments, the carrier barcode oligonucleotide contains from or about 10 to 30 nucleotides.

[0059] In some of any such embodiments, the one or more target polynucleotides comprise a polynucleotide of an immune molecule or chain thereof. In some embodiments, the one or more target polynucleotides comprise at least two target polynucleotides, each of which is an immune molecule chain polynucleotide.

[0060] In some of any such embodiments, the one or more target polynucleotides comprise one or more TCR polynucleotides or chains thereof. In some embodiments, the one or more target polynucleotides comprise a first T cell receptor (TCRα) polynucleotide and a second T cell receptor (TCRβ) polynucleotide. In some embodiments, the one or more target polynucleotides comprise a first polynucleotide belonging to the T cell receptor gamma (TCRγ) and a second polynucleotide belonging to the T cell receptor delta (TCRdelta).

[0061] In some of any such embodiments, the one or more target polynucleotides include one or more antibody polynucleotides or chains thereof. In some embodiments, the one or more target polynucleotides comprise a first immunoglobulin heavy chain (IgH) polynucleotide and a second immunoglobulin light chain (IgL) polynucleotide.

[0062] In some of any such embodiments, the barcoded polynucleotides are in the order (5' to 3'): first adapter, carrier barcode, molecular barcode, and second adapter. In some embodiments, the first adapter is located at or near the 5' region of the double-barcoded single-stranded polynucleotide. In some embodiments, the second adapter is located at or near the 3' region of the double-barcoded single-stranded polynucleotide.

[0063] Sequencing methods are provided, comprising sequencing one or more of a plurality of polynucleotides, such as barcoded polynucleotides (e.g., double barcoded polynucleotides) obtained by any method of the described embodiments or by any of the embodiments of the described polynucleotide libraries . In some examples, a transcriptome based on a plurality of polynucleotides, such as polynucleotide templates, is subjected to sequencing. In some cases, the method further comprises amplifying all or part of the transcriptome prior to sequencing. In some aspects, amplification is performed using a first primer set containing a first primer and a second primer specific to the first and second adapter sequences, respectively.

[0064] In some embodiments, one or more target polynucleotides are sequenced from a plurality of polynucleotide templates. In some instances, the method further comprises amplifying one or more target polynucleotide(s) from a plurality of polynucleotide templates prior to sequencing. In some examples, the full length sequence(s) of one or more target polynucleotides is amplified.

[0065] In some embodiments, the amplification is performed in the presence of a second primer set comprising one or more first primers that are complementary to one or more target polynucleotides and a second primer that is complementary to the first adapter sequence. In some cases, the second primer of the second primer set is complementary to the P7(C7) priming site, or a contiguous portion thereof, or the P5(C5) priming site, or a continuous portion thereof. In some aspects, the second primer of the second primer set is complementary to the P7(C7) priming site, or a contiguous portion thereof. In some embodiments, the second primer of the second primer set is or contains the sequence CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 39) or CAAGCAGAAGACGGCATACGAGAT [NNNNNN] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 28).

[0066] In some embodiments, one or more first primers that are complementary to one or more target polynucleotides are specific for the target sequence of an immune molecule or chain thereof. In some cases, the immune system molecule is a T cell receptor or an antibody. In some aspects, the one or more first primers are specific for the target sequence of the constant region of the immune molecule.

[0067] In some embodiments, the immune molecule is a TCR and the one or more first primers include AGTCTCTCAGCTGGTACACGG (SEQ ID NO: 37), ATGGCTCAAACACAGCGACCTC (SEQ ID NO: 38), or a combination thereof. In some embodiments, the implementation of the immune molecule is an antibody, and one or more of the first primers include any of SEQ ID NO: 29-36 or a combination thereof.

[0068] In some of any such embodiments, the method includes determining the cellular origin of one or more barcoded polynucleotides, optionally double barcoded polynucleotides. In some instances, determining cellular origin includes identifying the sequence information or sequences of double-barcoded polynucleotides that have the same carrier barcode, indicating their origin from the same cell.

[0069] In some of any such embodiments, the target polynucleotide is an immune molecule comprising a first polynucleotide strand and a second polynucleotide strand, and the method includes determining whether the first polynucleotide strand and the second polynucleotide strand correspond to the same cell in the presence of the same carrier barcode in double-barcoded sequenced polynucleotides. In some embodiments, the method further comprises quantifying or determining the number of polynucleotides with the same barcode, optionally the same molecular barcode and/or carrier barcode.

[0070] In some of any such embodiments, the plurality of barcoded polynucleotides are double-barcoded polynucleotides comprising a molecular barcode and a carrier barcode, and the method further includes identifying transcriptome sequences and target polynucleotide sequences, having the same carrier barcode, thereby identifying the transcriptomic information of the cell containing the target polynucleotide(s).

[0071] Transcriptome analysis methods are provided comprising (a) sequencing one or more target polynucleotides from a plurality of barcoded polynucleotides obtained by any of the described methods, or from a plurality of barcoded polynucleotides from any of the described polynucleotide libraries, wherein the barcoded polynucleotides are the code are double-barcoded polynucleotides containing a molecular barcode and a carrier barcode, thereby providing information about the sequence of a target polynucleotide from a plurality of cells; (b) sequencing all or part of the transcriptome derived from a plurality of barcoded polynucleotides obtained by any of the methods described, or from a plurality of barcoded polynucleotides from any of the described polynucleotide libraries, wherein the barcoded polynucleotides are double-barcoded polynucleotides. code and contain a molecular barcode and a carrier barcode, thereby providing transcriptome data from a plurality of cells; and (c) identifying sequence information from (a) and from (b) having the same carrier barcode as originating from the same cell.

[0072] Methods are provided for analyzing a selected single cell transcriptome, comprising (a) amplifying and sequencing one or more target polynucleotides from a plurality of barcoded polynucleotides obtained by any of the described methods or from a plurality of barcoded polynucleotides from any of the described polynucleotide libraries, wherein barcoded polynucleotides are double barcoded polynucleotides containing a molecular barcode and a carrier barcode, thereby providing sequence information for each of the target polynucleotides in at least one of a plurality of cells; (b) identifying the carrier barcode(s) associated with one of the target polynucleotides sequenced in (a), thereby identifying the selected individual cell carrying the target polynucleotide; (c) amplifying and sequencing the transcriptome, or a portion thereof, based on a plurality of cell barcoded polynucleotides bearing the carrier barcode identified in (b), thereby providing transcriptome data from the selected cell expressing the target polypeptide. In some embodiments, a transcriptome, or a portion thereof, from a selected cell is amplified or sequenced using a carrier barcode specific primer specified in (b) and a primer specific for a second barcode polynucleotide adapter sequence.

[0073] In some embodiments, the method includes matching the sequence information of a transcriptome, or a portion thereof, and at least one of the target polynucleotides derived from the same cell, wherein sequence information is determined from a plurality of barcoded polynucleotides derived from any from the described methods or from a plurality of barcoded polynucleotides of any of the described polynucleotide libraries, or determined by any of the described methods, where the barcoded polynucleotides are double barcoded polynucleotides containing a molecular barcode and a carrier barcode. In some cases, sequences having the same carrier barcode are grouped as belonging to the same cell. In some embodiments, the transcriptome data includes a parameter, characteristic, trait, or phenotype associated with cell function or activity. In some cases, transcriptome data is associated with cell activation, depletion, or proliferative activity.

Brief description of the drawings

[0074] In FIG. 1A is a diagram of the barcode stage: the exemplary method described here. The sketch illustrates a method for amplifying and barcoding two or more polynucleotides, such as a target polynucleotide, and one or more genomic or transcriptome polynucleotides or paired sequences, such as paired variable Ig (e.g., V _H and V _L mRNA) or TCR sequences ( for example, mRNA Vα/Vβ and Vγ/Vδ), for library preparation and sequencing of the immune repertoire (immune repertoire sequencing). Media Barcode (VB); Molecular barcode (MB). (Top) One drop (of many drops) of the emulsion is a typical vehicle containing one cell and other reaction components (eg, enzymes, buffers, oligonucleotides). (In the center). Exemplary methods for cell lysis and reverse transcription of RNA in a lysed cell using target-specific random and/or oligo-dT reverse transcription primers. (Bottom) The template switching step and molecular barcode (MB) labeling of individual molecules during the reverse transcription step.

[0075] In FIG. IB is a diagram of the amplification step of a typical method described here. The sketch illustrates a method for amplifying and barcoding two or more polynucleotides, such as a target polynucleotide, and one or more genomic or transcriptome polynucleotides or paired sequences, such as paired Ig variable (e.g., V _H and V _L mRNA) and TCR sequences ( for example, vRNA Vα/Vβ and Vγ/Vδ), for library preparation and immune repertoire sequencing. (Top) Independent carrier barcode (VB) amplification generates multiple copies identical to the VB in each drop. Molecular barcode (MB) cDNA molecules are simultaneously labeled with VB during the annealing and completion phases of the amplification cycle. (Center) Simultaneous amplification of double-barcoded cDNA molecules during an amplification cycle. (Bottom) Typical double-barcoded cDNA molecules ready for further processing (eg, purification, size selection, adapter ligation, amplification, and sequencing).

[0076] In FIG. 2 depicts the amplification and sequencing of double-barcoded transcripts by the typical methods described here. (Top) Amplification and sequencing of transcripts encoding a target gene of interest using a primer specific for the sequence of the universal primer of the first adapter and a primer specific for the target, with each primer associated with a sequencing adapter for sequencing. (Medium) Amplification and sequencing of all transcripts in the library, for example, a transcriptome or part of one or more cells, using primers specific for the universal priming sequence of the first adapter and the universal priming sequence of the second adapter. (Bottom) Amplification and sequencing of the transcriptome of a selected cell of interest, such as a cell determined to contain mRNA transcripts of the target gene of interest, using a primer specific for a particular carrier barcode (VB) common to all polynucleotides from that the same cell and primer specific for the universal priming sequence of the second adapter.

[0077] Figure 3 shows t-SNE plots for given single cell transcriptomes stained according to putative cluster identity according to Seurat software ( A ; dotted lines indicate clusters of events exhibiting predominantly the same color) or stained according to the type of full-length sequenced immune receptor, B-cell receptor (BCR), or T-cell receptor (TCR) ( B ; dotted lines indicate clusters of events showing predominantly the same color).

[0078] In FIG. 4 shows t-SNE plots of single cell transcriptome data such as those in FIG. 3 , stained to identify cells expressing designated sequences: Toll-like receptor 7 (TLR7 ; A ), CD3 glycoprotein epsilon chain on the surface of T cells (CD3E; B ), natural killer cell granular protein 7 (NKG7; C ) , C-Type 1 mannose receptor (MRC1; D )

Detailed description

[0079] Provided herein are methods and compositions for analyzing gene expression in single cells or in multiple single cells. The proposed methods make it possible to efficiently generate a plurality of high-quality polynucleotides (for example, DNA) belonging to a single cell, to perform sequencing of libraries containing polynucleotides of the entire transcriptome or part thereof, and one or more full-length polynucleotide sequences of a target gene of interest, such as a full-length product of a paired immune receptor , for example TCR or antibodies, whereby the target polynucleotide(s) and polynucleotides from the full or partial transcriptome derived from the same cell can be identified. In some embodiments, each of the plurality of polynucleotides in the library contains adapter sequences (eg, a first adapter and a second adapter) that allow NGS sequencing of all recovered products, as opposed to sequencing specific genes that must be selected during the course of the experiment. Thus, in some aspects of the provided method, subsequent PCR amplification may be carried out using primers specific for these adapter sequences and/or primers specific for the target polynucleotides of interest and/or cell specific primers. In some embodiments, the provided methods allow processing tens or hundreds of thousands of cells in a single experiment, thereby obtaining single cell sequencing data, e.g., RNA sequencing data, such as mRNA count, in combination with full-length sequences of immune molecules, e.g., antibodies. or TCR, in an efficient and high performance way.

[0080] In some cases, direct sequencing of all or partial genome content and mRNA in tissue is increasingly being used to enable the analysis of alternative splicing, regulatory/promoter regions, and polyadenylation signals without the need for prior selection of previously known genes (Cloonan et al., Nat Methods 5 (7): 613-9 (2008)). However, current methods for analyzing the content of mRNA in cells by direct sequencing are based on the analysis of total mRNA obtained from tissue samples, usually containing millions of cells. This means that much of the functional information present in individual cells is lost or blurred when gene expression is analyzed in total mRNA. In addition, gene expression during dynamic processes such as the cell cycle is difficult to observe as a population average. Therefore, for some applications, single-cell based direct sequencing approaches are preferable to bulk sample operation.

[0081] In some cases, it is desirable to analyze the content of the genome or mRNA in a selected cell, such as a cell expressing a particular gene or genes of interest, such as target genes. In some cases, it is desirable to determine the genomic or transcriptome content of a selected cell, as well as obtain the full length sequence of a target gene, such as an immune receptor. Existing single cell transcriptome sequencing tools include microarrays, 96-well methods such as traditional FACS well sorting, and microfluidic tools such as Fluidigm C1. These tools can be used to prepare full length transcriptome and target libraries, but their throughput is limited because they are limited in the number of cells available for analysis (eg hundreds to thousands of cells).

[0082] In some cases, it is desirable to analyze the content of the genome or mRNA in a selected cell, such as a cell expressing a particular gene or genes of interest, such as target genes (eg, immune receptor). Ultra-high throughput methods using microwell arrays or emulsions have been described that allow single cell transcriptome sequencing (see, for example, Klein et al., Cell (2015) 161 (5): 1187-1201; Macosko et al., Cell ( 2015).) 161 (5): 1202-1214; WO/2015/164212; WO/2016/040476), but effective capture of full-length target sequences, such as full-length immune receptor sequences, is not possible with current technology. These methods are also limited to smaller numbers of cells, typically a few thousand, due to limitations imposed by the bead-based approach, which requires a larger droplet and larger reaction volume per cell.

[0083] In embodiments of the provided methods, target sequences, such as immune target molecules (e.g., antibody or TCR), and multiple cell genome or transcriptome sequences are produced in one simultaneous reaction and provide a mechanism for combining sequence information for all sequences. originating from the same cell. In some aspects, the currently disclosed methods, in combination with high-throughput sequencing technology, allow the analysis of a large number of individual cells and analysis in a single reaction. In principle, any number of cells can be sequenced with any number of target regions per cell. In some aspects, the number of individual cells that can be processed is only limited by practical limitations such as the speed of high throughput sequencing. In some embodiments, the implementation of the methods disclosed in this document are adaptable to the use of granules. In other embodiments, the methods disclosed herein do not include a bead-based sequencing or amplification step.

[0084] In some aspects, the proposed methods overcome or reduce the problems of existing methods by providing a method for obtaining cDNA libraries that can be used to analyze gene expression in a variety of individual cells. In some embodiments, the provided methods result in an ultra-high throughput polynucleotide sequencing library that allows reproduction of full-length target sequences ready for synthesis, including sequences of paired heterodimeric or multimeric targets, while capturing additional quantitative genomic or transcriptomic information about cells identified as expressing this(s) sequence(s)-target(s).

[0085] In particular, the provided methods are for obtaining a polynucleotide library, such as a cDNA library, from a plurality of single cells. The methods are based on determining the levels of gene expression in a population of individual cells, which can be used to identify natural changes in gene expression at the cellular level. The methods can also be used to identify and characterize the cellular composition of a cell population, even in the absence of a suitable cell surface marker. The methods described herein also provide the advantage of generating a cDNA library representing RNA in a cell population using single cells, whereas classically prepared cDNA libraries typically require total RNA isolated from a large population. Thus, in some aspects, a cDNA library produced using the provided methods allows at least a reliable representation of the RNA content of a population of cells using a smaller subpopulation of individual cells, along with additional benefits as described herein.

[0086] The present embodiments are based on single cell target sequencing emulsion methods such as immune target sequencing and transcriptome analysis. In some aspect, cells from a sample containing a population of cells are encapsulated in a separate carrier (eg, drops), for example, using microfluidic emulsion based methods. Methods are then carried out for attaching specific carrier barcodes (e.g., droplets) to target polynucleotides (e.g., target mRNA transcript amplicons) and/or to multiple polynucleotides (e.g., full or partial transcriptome mRNA transcript amplicons) in the emulsion droplets, allowing perform high-throughput genetic analysis and/or analysis of the expression of individual cells contained in the drops. In some aspects, the carrier barcodes are initially present as separate DNA templates with a randomized region of the central sequence flanked by known primer sites. Templates can be reverse transcribed to generate an amplicon and/or amplified by PCR to produce one or more amplicons within a carrier, such as drops, and can be fused to nucleic acids of cellular origin by overlapping sequences. In some aspects of the method, a nucleic acid of cellular origin is amplified using a gene-specific PCR primer to amplify a target gene or genes of interest, such as the gene for an immune molecule such as an antibody or T cell receptor (TCR). In some cases, the ability to amplify multiple target genes can provide information about various features of the cell, such as phenotype, activity, or other features of the cell.

[0087] In some cases, the addition of additional target genes can link information about a particular immune molecule, such as TCR, to information about the cellular phenotype of the cell. In other aspects of the method, full-length transcriptome nucleic acids of cellular origin can be amplified using primers that allow sequencing of the entire or partial transcriptome of the cell, and transcriptomes derived from the same cell can be grouped. In some aspects, random reverse transcription primers are used to generate amplicons corresponding to transcriptome transcripts. In some embodiments, a universal priming site can be added to the ends of barcoded polynucleotides, such as in the added first and second adapters, so that the entire library can be amplified and sequenced in its entirety by a high throughput shotgun method.

[0088] In some embodiments, a nucleic acid amplification reaction of cellular origin can be carried out by reactions within a single carrier ("in one tube") in which a) cell lysis is performed; b) reverse transcription of the target mRNA; c) molecular barcoding of each cDNA; d) PCR amplification of the sample-specific DNA barcode; and e) attaching a copy of the carrier barcode to each cDNA. In some embodiments, the products can be reproduced and sequenced, for example, using any of a variety of sequencing platforms. For example, you can use the Illumina MiSeq platform, using 325x300bp to sequence the entire length of each product. In some aspects, barcode carriers (droplets) allow you to identify all the products of each individual cell. In certain aspects, molecular barcodes can quantify per-cell expression and, in some cases, eliminate sequencing and RT-PCR errors.

[0089] In the embodiments shown, the transcriptome sequencing process can be performed separately from the target sequencing of multiple selected transcripts, such as TCRs, since the amplified library can be processed separately. This makes it possible to apply the method several times on separate aliquots of the same amplified barcoded library. With the provided methods, many useful approaches can be implemented due to the possibility of performing different experiments at different times on the same amplified barcoded library. In one example, the methods can be used to first target sequencing of a target molecule of interest, such as TCR, in a sample, which can lead to the identification of several specific cells of interest. In some aspects, PCR or "capture" (capture) oligonucleotides can then be designed to target barcodes of carriers belonging to cells of interest, allowing capture and sequencing of all barcoded transcripts of only these cells. In some aspects, this significantly reduces the cost of sequencing the entire library.

[0090] In certain aspects, the methods and compositions described herein are advantageous for analysis of single cells, for example, analysis aimed at studying genomes, transcriptomes, proteomes, metabolic pathways, and the like in complex cellular samples. The analysis of multiple cells in heterogeneous cell populations is particularly advantageous when studying complex samples or mixtures. Complex samples or mixtures of cells include, for example, peripheral blood mononuclear cell (PBMC) samples, metagenomic samples, normal and cancer tissue sections, embryonic and stem cell colonies. Genome and transcriptome sequencing is desirable to identify divergent cell types or to study cells at specific stages, such as various stages of activation, depletion, or proliferation. Specific applications include molecular T and B cell profiling, haplotyping and HLA typing.

[0091] "Metagenomic samples" refers to samples containing genomes from different sources, such as species. For example, the present approach can be applied to mixtures of bacteria of different species to allow sequencing of nucleic acids from multiple bacteria in a single assay, followed by correlation of the sequences to the same bacterial cell. Similarly, nucleic acid sequences of one cell type found among cells of another cell type, such as immune cells infiltrating a tumor. In such cases, tumor-infiltrating immune cell nucleic acids can be mapped to the full-length immune receptor sequence(s) or binding fragment thereof using the methods presented herein.

[0092] Embodiments of the provided methods also allow the creation of carriers for a large number of single cells. Using the similarity of expression profiles, it is possible to construct a cell map showing how these cells are related to each other. This map can be used to distinguish cell type in silico when clusters of closely related cells are found. By creating carriers not of a few cells, but of a large number of individual cells, one can use the similarity of expression profiles to build a map of cells and their relationships. This method allows access to direct data on the expression of each individual cell type present in the population without the need for prior purification/isolation of that cell type. In addition, if known markers are available, they can be used in silico to determine the position of cells of interest.

[0093] The present embodiments include a method for obtaining a polynucleotide library, such as a cDNA library, from a plurality of individual cells by releasing mRNA from each individual cell to obtain a plurality of individual samples, wherein the mRNA in each individual mRNA sample is taken from a single cell, followed by synthesizing a first strand cDNA (i.e., amplicon) per mRNA in each individual mRNA sample and incorporating a nucleotide barcode (e.g., molecular barcode and/or carrier barcode) into the cDNA amplicon to generate multiple barcoded cDNA samples ( e.g., double-barcoded) (in other words, where the cDNA in each barcoded cDNA sample, the complementary mRNA of a single cell, is combined with other barcoded cDNA, followed by amplification of the combined cDNA samples to create a cDNA library containing double-stranded cDNA with barcode). In some embodiments, the resulting amplicon is a double-barcoded single-stranded cDNA, such as double-barcoded single-stranded cDNA, corresponding to one or more mRNA templates. In some embodiments, the barcoded double strand cDNA is denatured to generate barcoded single strand cDNA to facilitate the addition of an adapter, such as a sequencing adapter. Using the above method, it is quite possible to prepare samples for sequencing from several hundred individual cells in a short time. Conventional methods for obtaining a library of RNA fragments for sequencing include gel removal steps that are labor intensive. In some aspects of the methods described herein, many cells are obtained as a single sample (after cDNA synthesis), which makes it possible to prepare a large number, for example several hundred, cells for sequencing. In addition, technical variations can be minimized as each sample of multiple cells is prepared together (in the same tube).

[0094] In some aspects of the invention, each cDNA sample obtained from an individual cell is labeled with a barcode that allows analysis of gene expression at the individual cell level. This allows the study of expression during dynamic processes such as the cell cycle and the analysis of different cell types in complex tissue (such as the brain). In some aspects, cDNA samples may be pooled prior to analysis. Sample pooling simplifies sample processing from each individual cell and reduces the time required to analyze single cell gene expression, allowing for high throughput gene expression analysis. Pooling cDNA samples prior to amplification also provides the advantage that technical differences between samples are virtually eliminated. In addition, because cDNA samples are pooled prior to amplification, fewer amplification reactions are required to generate enough cDNA for subsequent analysis compared to amplifying and processing cDNA samples from each individual cell individually. This reduces amplification errors and also means that any amplification bias to one side or the other will be the same for all cells used to obtain pooled cDNA samples. RNA purification, storage, and handling are also not required, helping to eliminate problems caused by the unstable nature of RNA.

[0095] T cell receptor chain pairs and immunoglobulin antibody chain pairs are among the types of immune receptors that are expected to be sequenced using currently disclosed methods. In some embodiments, the provided methods allow the generation of polynucleotide libraries for high-throughput sequencing and analysis of gene expression, comprising sequences of one or more target sequences, such as one or more immune receptor sequences, and sequences that can be combined to obtain genomic and/or transcriptome sequencing information. In some embodiments, a polynucleotide library can be created, which is a human-derived panel library used to detect antibodies and/or TCRs in a patient or group (cohort) of patients with specific common features. In some embodiments of the provided method, the starting material may be any source containing a population of cells of interest, which in turn contains or may contain a target polynucleotide, such as an immune molecule or receptor, such as an antibody or TCR. In some embodiments, the starting material may be a peripheral blood or tissue biopsy from which immune cells are readily isolated or subdivided into naïve cells, memory cells, and/or antibody-secreting (ASC) cells, if desired. In some embodiments, the implementation of the proposed method can be applied to many different types of single or paired variable sequences, for example, pairs of T-cell receptor chains and pairs of immunoglobulin antibody chains.

[0096] In some aspects, the cDNA libraries obtained using the provided methods are suitable for the analysis of individual cell gene expression profiles by direct sequencing, that is, these libraries can be used to study gene expression, including the expression of genes associated with a particular target polynucleotide representing of interest, or cells carrying a polynucleotide such as, for example, an immune system molecule or a receptor, such as an antigen, antibody, or TCR. In some embodiments, those gene expression profiles that were not previously known can be analyzed. In some embodiments, the provided methods can be used to characterize or compare the transcriptional state of each of a plurality of cells from a sample, such as an activated, depleted, proliferating state, or according to another desired cell parameter or attribute. In some embodiments, the provided methods can be used to facilitate the discovery of molecules with therapeutic potential, such as TCRs, by analyzing the response of particular cells bearing a particular TCR specific for an antigen of interest. In some embodiments of the provided methods, cells expressing a TCR associated with a desired cellular response, such as a natural one, or degree of T cell activation, can be identified. In some embodiments, the methods provided allow a more representative set of data to be collected by analyzing the entire transcriptome, as opposed to existing methods that require pre-existing knowledge and/or selection of a smaller set of candidate genes.

I. Polynucleotide library for targeted and transcriptomic analysis

[0097] In some embodiments, the methods provided herein are directed to the amplification and sequencing of one or more target polynucleotide molecules, and to the amplification and sequencing of a collection of polynucleotides, such as one or more target molecules, and a collection of polynucleotides from a single cell. or population of cells. In some embodiments, the methods and compositions described herein may be applicable to the analysis of single cells, for example, to study genomes, transcriptomes, proteomes, metabolic pathways, and the like in complex cell samples. In other aspects, the methods and compositions described herein can be used to detect immune receptors, for example, by matching heavy and light chain immunoglobulin or T cell receptors on individual B and T cells, as well as for HLA typing. In other embodiments, antibody pairing information and single cell analysis can be combined to link information about cell function or cell status to expression of an identified immune receptor sequence. In other aspects, the methods and compositions described herein can be used to monitor the impact and effect of small molecules and drugs or immunotherapy in complex normal or cancer samples for diagnosis or the discovery of new drugs or treatment regimens. In yet another embodiment, the methods and compositions can be used to detect and analyze target pathogens such as bacteria or viruses in biological samples.

[0098] In some embodiments, the provided methods may include various features of the methods described in International Publication Nos. WO2012/048341, WO2014/144495, WO2016/044227, WO2016/176322 or WO2017/053902, each of which is included in its entirety as a reference.

[0099] The present invention utilizes steps in which nucleic acids are manipulated to create polynucleotide libraries for sequencing. In some embodiments, implementation of the present invention uses the stage, which manipulate nucleic acids to obtain polynucleotide target molecules, including sequences containing variable regions of an immune receptor, such as an antibody or TCR produced by an immune cell. In some instances, the target polynucleotide molecule is a recombinant monoclonal antibody molecule. In some embodiments of the present invention, steps are used in which nucleic acids are manipulated to obtain polynucleotides representing the transcriptome or genome of one or more cells. In general, in some embodiments of the invention, in order to amplify the genetic materials of a cell, such as an immune cell and/or a T cell, reverse transcription polymerase chain reaction (RT-PCR; reverse transcription-PCR) is used to perform cDNA amplification of the genetic material. immune cell.

[0100] In some embodiments, the methods can be used to obtain sequence information of a target polynucleotide of interest, such as a TCR or an antibody, within a cell. Target genes can be obtained from the genomic DNA or mRNA of a cell from a sample or population of cells. The sample or population of cells may include immune cells. For example, for antibody target molecules, immunoglobulin genes can be obtained from the genomic DNA or mRNA of immune cells or T cells. RNA can be heavy chain (V, D, J segments) or light chain (V, J segments). In some embodiments, the starting material is an RNA derived from immune cells and consisting of V, D, J gene segments encoding an antibody and containing a constant region.

[0101] In some embodiments, in addition to obtaining full-length sequence data of a target polynucleotide of interest, such as an immune molecule such as an antibody or TCR, the provided methods also allow efficient generation of high quality DNA libraries for sequencing. both on the basis of a complete transcriptome product and on the basis of a full-length target, which includes a multi-subunit target, polynucleotide(s), such as antibodies or TCRs, containing full-length products of paired immune receptors.

[0102] In some embodiments, such methods include adding (eg, by ligation) an adapter DNA sequence to single-stranded polynucleotide products, which may allow amplification and next generation sequencing (NGS) of a single cell or multiple single cell transcriptome.

A. Polynucleotide Libraries

[0103] A library obtained in accordance with the methods described herein may be a library containing a target sequence, full length or significant length, such as an antibody or TCR sequence, with appropriate barcodes, such as a carrier barcode and molecular barcodes. In some embodiments, the library contains a significant or full length target sequence, e.g., an antibody or TCR sequence that includes both antibody or TCR chains, and sequences corresponding to one or more partial or complete cell transcriptome transcripts based on the original target sequence, e.g., antibodies or TCR, each with a carrier barcode and a molecular barcode. In such embodiments, a target sequence, of significant or full length, e.g., an antibody or TCR sequence, and sequence(s) corresponding to one or more partial or complete transcriptome transcriptomes of the cell from which the target sequence is derived, e.g., an antibody or TCR, contain the same carrier barcode and also contain a molecular barcode that is unique for each original transcriptome transcript. In some aspects, the carrier barcode is included in a first adapter that is attached to each target polynucleotide as well as to each polynucleotide of a set of polynucleotides representing transcriptome transcripts.

[0104] In some embodiments, methods are provided for obtaining a library of polynucleotides, wherein an adapter (hereinafter also referred to as a second adapter) is added to each barcoded single-stranded polynucleotide from the set labeled with the previous or first adapter such that the adapters are at opposite ends of the polynucleotide , and where the set of single-stranded polynucleotides with a barcode includes (i) one or more single-stranded polynucleotide(s) target(s) that are complementary to one or more target polynucleotides present in a cell belonging to a population of cells; and (ii) a collection of single-stranded polynucleotides, each of which is complementary to a polynucleotide in a cell, where each of the plurality of barcoded single-stranded polynucleotides contains a carrier barcode that is the same for all complementary polynucleotides from the same cell belonging to the cell population. The adapter, such as each of the first and second adapters, contains a universal priming sequence that can be used to amplify or sequence adapter-tagged double-barcoded polynucleotides.

[0105] In some embodiments, a library of polynucleotides may be sequenced. In some embodiments, a library created in accordance with the methods described herein may contain appropriate clustering segments for sequencing. In some embodiments, multiple copies of identical molecular barcodes can be generated. In some embodiments, multiple copies of polynucleotides containing identical molecular barcodes can be created for each original unique target polynucleotide molecule. In some embodiments, multiple copies of polynucleotides containing identical molecular barcodes can be generated for each original unique target polynucleotide molecule labeled with a carrier barcode. Any or all of the sequences can be sequenced and assembled, for example, to determine the complete or partial transcriptome of the cell expressing the target sequence(s).

[0106] The starting material may be RNA or DNA from a cell, such as immune cells, or T cells. In some instances, the cell may be a cell known or suspected to contain the desired target polynucleotide, such as an immune receptor such as a TCR, or an antibody. For example, in the case of an antibody, the target cell is a cell containing the V, D, J gene segments encoding the antibody and includes a constant region. In some embodiments, the target polynucleotide comprises heavy chain segments (V, D, J segments) or light chain segments (V, J segments).

[0107] The original polynucleotide material, such as RNA, can be reverse transcribed into cDNA using a single polynucleotide or the whole pool. Exemplary primers in the pool of polynucleotides for reverse transcription of a target polynucleotide may contain a portion complementary to a region of the target polynucleotide and/or may include sequences for reverse transcription of all or part of the transcriptome. In some instances, polynucleotides may include a region complementary to that of a target RNA, such as a target constant region or an mRNA poly-A tail. In some instances, multiple oligonucleotides, such as primers, may be used to anneal one or more constant region target sequences. In some aspects, one or more polynucleotides include specific, polydT and/or random hexamer primers.

[0108] Reverse transcriptase can be used to carry out the reverse transcription reaction. In specific embodiments, the reverse transcriptase may have non-template terminal transferase activity. When a reverse transcriptase having non-template terminal transferase activity reaches the end of the template, it may add three or more non-template residues, for example, three or more non-template cytosine residues. In some embodiments, Superscript II™ reverse transcriptase is used for this purpose. In some embodiments, Maxima™ reverse transcriptase is used for this purpose. In some embodiments, Protoscript II™ reverse transcriptase is used for this purpose. In some embodiments, Maloney murine leukemia virus (MMLV-RT) reverse transcriptase is used for this purpose. In some embodiments, HighScriber™ reverse transcriptase is used for this purpose. In some embodiments, a terminal deoxynucleotidyl transferase is used for this purpose. In some embodiments, avian myeloblastosis virus (AMV) reverse transcriptase is used for this purpose. Any reverse transcriptase capable of transcribing RNA and having non-template terminal transferase activity can be used. Also, any reverse polymerase capable of transcribing RNA and having non-template terminal transferase activity can be used. Any reverse polymerase capable of transcribing DNA having non-template terminal transferase activity can be used. cDNA resulting from reverse transcription may be labeled with one or more barcodes. In some examples, cDNA resulting from reverse transcription may be labeled with a carrier barcode and a molecular barcode. Various oligonucleotides of a particular design can be used for barcode labeling.

[0109] In some embodiments, template switching can be used to create libraries for, for example, immune repertoire sequencing and/or transcriptome analysis. For example, template switching can be used during reverse transcription to generate a reverse transcription product region that is complementary to a polynucleotide carrying a barcode, such as a carrier barcode or carrier molecular barcode. Template switching can be used during reverse transcription to eliminate problems associated with PCR. These methods can be used to sequence antibodies, for example, using a high throughput sequencing platform.

[0110] In some embodiments, the carrier barcode includes a region of randomized sequence flanked by known primer sites. For example, cDNA may be labeled with a carrier barcode comprising a region of ~20 degenerate nucleotides with/without one or more defined intercalating bases, such as NNNNWNNNNWNNNNWNNNNW (SEQ ID NO: 99; where N is any nucleotide and W is a specific intercalating base, representing A or T), or NNNNWISCNNNNWISCNNNN (SEQ ID NO: 100; where N is any nucleotide; W is a specific intercalating base representing A or T; I is a specific intercalating base representing A, T, G or C ( i.e. N); and where S is an intercalating base which is G or C; and C is a specific intercalating cytosine). Other exemplary sequences included in carrier barcode oligonucleotides include NNNNWNNNNWNNNN (SEQ ID NO:80), WNNNNWNNNNWNNNN (SEQ ID NO:81), NWNNNWNNNNWNNNN (SEQ ID NO:82), or NNWNNNNWNNNNWNNNN (SEQ ID NO:83), where N is any nucleotide and W is adenine or thymine. The carrier barcode may also include known primer sites that can be recognized by forward and reverse primers to amplify carrier barcodes in the reaction mixture prior to their attachment to transcripts or labeling, for example during annealing. Examples of primers with a carrier barcode are shown in SEQ ID NO: 4 and SEQ ID NO: 5 or SEQ ID NO: 10 and SEQ ID NO: 11. In some cases, a collection of carrier barcodes containing the same primer regions but base-shifted in the degenerate portion, two or more, represented by any of SEQ ID NOs: 80, 81, 83, 99, or 100, is added to the carrier to produce base-shifted labeled polynucleotide products to increase product diversity in the sequencing process. In some embodiments, the oligonucleotide containing the carrier barcode is part of an adapter, such as a first adapter containing a universal primer site (eg, P7). The first adapter, as described herein, may include a universal priming site and a carrier barcode. Examples of such oligonucleotides containing carrier barcodes, including the universal priming site, degenerate portion, and primers are shown in SEQ ID NOs: 2, 6, 7, 8, or 9, or are a pool of any two or more of SEQ ID NOs: 6, 7 , 8, or 9. In some cases, a carrier barcode or pool of carrier barcodes can be used to label cDNA molecules processed in a single carrier. In specific examples, cDNA molecules processed in the same carrier are complementary to RNA molecules of the same cell.

[0111] In some embodiments, the molecular barcode includes a degenerate sequence to uniquely label polynucleotide transcripts in a reverse transcription reaction. In some aspects, the molecular barcode is part of a template switch oligonucleotide, wherein the template switch oligonucleotide includes template sequences for reverse transcriptase, and thus the molecular barcode is included in each complementary polynucleotide. In some embodiments, a template switch oligonucleotide may comprise (1) a 5' end region complementary to a 3' tagged first adapter polynucleotide containing a carrier barcode, (2) a molecular barcode, and (3) a 3' portion complementary to 3 '-end of the ledge. In some embodiments, a template switch molecule, such as a template switch oligonucleotide containing a barcode (eg, a molecular barcode), may include modified bases to minimize artifacts. An exemplary template switch oligonucleotide containing an exemplary molecular barcode is shown in SEQ ID NO: 3.

[0112] Reverse transcription reactions such as those described above can be carried out in the presence of a 3' labeled (labeled) polynucleotide. A 3'-tagged polynucleotide can be a polynucleotide used to add nucleic acids to the 3' end of a selected polynucleotide, such as a target cDNA, or to a polynucleotide (eg, cDNA) complementary to a cellular transcriptome transcript. The 3'-tagged polynucleotide may be a polynucleotide used as a template for completing nucleic acids at the 3' end of a target polynucleotide, for example, in a cDNA. A 3'-tagged polynucleotide may be a polynucleotide that hybridizes to the 3' end of a target polynucleotide, such as cDNA. A 3'-tagged polynucleotide may be a polynucleotide containing a 3' region, such as a 3' region hybridizing to the 3' end of a target polynucleotide, eg cDNA. For example, a 3'-tagged polynucleotide may contain a segment, such as a segment that anneals to three or more non-template residues. In some embodiments, the 3'-labeled polynucleotide is a polynucleotide bearing a molecular barcode. In some embodiments, the implementation of the 3'-tagged polynucleotide may contain a molecular barcode. In some embodiments, the 3'-tagged polynucleotide may contain 3'-riboguanosine residues or analogues thereof at the 3' end (rGrGrG) (i.e., RNA bases) that are complementary and can anneal to a strand produced by a reverse transcription enzyme ( for example, the sequence CCC). In some embodiments, three or more guanine residues can be used in place of riboguanosine residues (ie, DNA nucleotides instead of RNA nucleotides). In some embodiments, the 3'-tagged polynucleotide may contain 1 or 2 riboguanosine residues at the 3' end and a riboguanosine residue or analogue thereof at the 3' end (rGrGG) that are complementary and applicable to anneal the chain resulting from the operation of the enzyme reverse transcription (eg CCC sequence).

[0113] After annealing a 3'-labeled/labeled polynucleotide with a CCC cDNA strand, reverse transcriptase can continue to extend the cDNA along the length of the labeled polynucleotide, thereby adding a molecular barcode or its complement to the target population of polynucleotides, such as cDNA, during the reaction . For example, a 3'-tagged polynucleotide can be a polynucleotide containing a 5' to 3' region that hybridizes to the 3' end of the target polynucleotide. The 5' to 3' region hybridizing to the 3' end of the target polynucleotide may contain a region that is not complementary to the target polynucleotide represented by the cDNA. The 5' to 3' region hybridizing to the 3' end of the target polynucleotide may contain a molecular barcode. The 5' to 3' region hybridizing to the 3' end of the target polynucleotide may contain a region complementary to the polynucleotide with a carrier barcode, or be complementary to a polynucleotide complementary to that. In other experiments, matrix switching can be done in separate reactions. For example, a 3' labeled polynucleotide may be added after the reverse transcription reaction and enzymes such as reverse transcriptase or polymerase may be used to complete the labeled polynucleotide. Since a labeled polynucleotide may contain a unique degenerate molecular barcode on each carrier molecule, each carrier cDNA may be individually labeled/labeled with a molecular barcode. In some embodiments, the template switch may be performed simultaneously with the reverse transcription reaction.

[0114] In some embodiments, a 3'-tagged polynucleotide, such as a molecular barcode polynucleotide, may further comprise a 5'-region, such as a 5'-terminal region, complementary to or complementary to the 3'-tagged polynucleotide and contain another a barcode, such as a media barcode. In some embodiments, a target polynucleotide containing a molecular barcode or a polynucleotide complementary to one, such as a tagged cDNA molecule, may contain a 3' region, such as a 3' end region, that is complementary to or complementary to the 3' tagged polynucleotide , and containing another barcode, such as the carrier's barcode.

[0115] In some embodiments, the 3'-tagged polynucleotide is a carrier-coded polynucleotide. After generating a polynucleotide containing a molecular barcode or its complement from a target polynucleotide, a carrier barcode can be added to the target polynucleotide with a molecular barcode. A 3'-tagged polynucleotide can be a polynucleotide used to extend nucleic acids towards the 3' end of a target polynucleotide, such as a molecular barcode target polynucleotide. The 3'-tagged polynucleotide can be a polynucleotide used as a template for extending nucleic acids towards the 3' end of a target polynucleotide, such as a molecular barcoded target polynucleotide. A 3'-tagged polynucleotide may be a polynucleotide that hybridizes to the 3' end of a target polynucleotide, such as a molecular barcoded target polynucleotide. A 3'-tagged polynucleotide can be a polynucleotide that contains a 3' region, such as a 3' end region, that hybridizes to the 3' end of a target polynucleotide, such as a molecular barcoded target polynucleotide. The carrier barcode polynucleotide may comprise a 3' region, such as a 3' end region, that hybridizes to the 3' end of the target polynucleotide having the molecular barcode.

[0116] After annealing a 3'-tagged polynucleotide to a target polynucleotide having a molecular barcode, reverse transcriptase can continue to extend the cDNA towards the 3'-tagged polynucleotide, such as the polynucleotide with a carrier barcode, thereby attaching the barcode the carrier or its complement to the target population of polynucleotides, such as target polynucleotides with a molecular barcode, during the reaction. For example, a 3'-tagged polynucleotide can be a polynucleotide containing a 5' to 3' region that hybridizes to the 3' end of the target molecular barcode polynucleotide. The 5' to 3' region hybridizing to the 3' end of the target molecular barcode polynucleotide may contain a region that is not complementary to the target or molecular barcode polynucleotide. The 5' to 3' region that hybridizes to the 3' end of the target molecular barcode polynucleotide may contain a carrier barcode.

[0117] In some embodiments, the implementation of the 3'-tagged polynucleotide is an amplification product. In some embodiments, the 3' labeled polynucleotide is a single molecule amplification product. In some embodiments, the 3' labeled polynucleotide is an amplification product of a carrier barcoded polynucleotide. In some embodiments, the 3'-labeled polynucleotide is an amplification product of a single carrier-coded polynucleotide. The region located in the 5' to 3' region that hybridizes to the 3' end of the target molecular barcode polynucleotide may contain a region complementary to the primer or its complement. The 5'-3' portion of the region that hybridizes to the 3' end of the molecular barcode target polynucleotide may contain a region complementary to the primer or its complement that can be used to amplify the polynucleotide carrying the carrier barcode.

[0118] In some embodiments, the 3'-labeled polynucleotide can act as a primer, such as a forward primer, to amplify a cDNA containing a molecular barcode, and the DNA polymerase is able to extend the sequence to form a single-stranded, double-barcoded polynucleotide molecule. a code that is complementary to the cDNA and contains the carrier barcode, the molecular barcode, and the coding sequence of the target gene or transcript. In some embodiments, the double-barcoded single-stranded polynucleotide molecule comprises, in the 5' to 3' direction: a carrier barcode, a molecular barcode, a target gene or transcript coding sequence, and a first adapter. In some embodiments, the oligonucleotide containing the carrier barcode contains a second adapter, and the double-barcoded single-stranded polynucleotide molecule contains in the 5' to 3' direction: the second adapter, the carrier barcode, the molecular barcode, the coding sequence for the target gene or transcript and the first adapter.

[0119] Labeled cDNA resulting from reverse transcription can be amplified one or more times, for example, using PCR amplification. For amplification, you can use different primers of a certain design. The product of a first amplification reaction, such as PCR, can be amplified using a second amplification reaction, such as a first or second phase PCR. Various primers can be used for the amplification step. An amplified polynucleotide library can be generated using the methods described herein. In some examples, the resulting library may contain the entire or partial sequence of an antibody or TCR with appropriate molecular and carrier barcodes. The library may also contain sequences corresponding to one or more partial or complete transcriptome transcripts with corresponding molecular and carrier barcodes.

[0120] A double-barcoded target polynucleotide, such as a cDNA containing a molecular barcode and a carrier barcode, can then be amplified, for example, by PCR. PCR can be carried out, for example, using a set of primers. The product of the above PCR reaction can then either be amplified one or more times, for example during one or more PCR cycles, or directly sequenced. In some embodiments, the primer set may include forward and/or reverse primer(s) specific to the first and/or second adapter. In some embodiments, the primer set may include an oligonucleotide containing the carrier barcode as a forward primer and a reverse primer specific to the first adapter. In some embodiments, the primer set may include primer sets that include forward and reverse primers that bind the second and first adapters, and an oligonucleotide containing the carrier barcode as an additional primer, e.g., as an additional forward primer. Primer examples are described in the Examples here.

[0121] When sequencing, sequences with the same molecular barcodes can be grouped or paired. When sequencing, sequences with the same carrier barcodes can be grouped or paired. When sequencing, sequences with identical target sequences can be grouped or paired. In some embodiments, sequence reads may be combined into consensus sequences. Folding clustered or paired sequential reads into a consensus consensus sequence can thus reduce or eliminate sequencing and PCR errors. Sequencing can be performed using the first primer site for the first read. Sequencing can be performed using the first primer site for a second read. Sequencing can be performed using the second primer site for a second read.

[0122] In some embodiments, immune receptor chains, such as TCR chains or antibodies containing the same carrier barcodes, can be paired. In some cases, heavy and light chains of antibodies containing the same carrier barcodes can be paired. In some embodiments, the paired strands can be cloned into a mammalian cell vector system. An immune receptor construct, such as an antibody, may be expressed in other human or mammalian host cell lines. This construct can then be verified by transient transfection and Western blotting of the expressed antibody or TCR of interest.

[0123] In some aspects, the invention provides a method for generating a library of heavy and light chain antibody sequences and/or alpha and beta chain TCR sequences and/or gamma and delta chain TCR sequences, comprising generating a plurality of nucleic acid constructs with unique streaks. codes, where each construct includes a unique N-mer and a functional N-mer. The functional N-mer may be a random N-mer, PCR primer, universal primer, antibody, sticky end, or any other sequence. The method may include creating M sets, each including N fluid compartments, each containing one or more copies of the unique construct. With this method, barcode libraries of higher complexity can be created by adding an additional construct to each compartment in a set and repeating it for each set, so as to create M compartments, each containing a unique pair of constructs. These pairs can be hybridized or ligated to generate new constructs. Within each construct in the barcode library, each unique N-mer can be prepared for identification by sequencing, carrier probe hybridization, other methods, or a combination of methods.

[0124] Methods for amplifying RNA or DNA are well known and can be used in accordance with the present invention, based on the instructions and guidelines provided here, without undue experimentation. Known methods for amplifying DNA or RNA include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, for example, US Pat. and 4,921,794 (Tabor, et al); 5,142,033 (Innis); 5,122,464 (Wilson, et al.); 5,091,310 (Innis); 5,066,584 (Gyllensten, et al); 4,889,818 (Gelfand, et al.); al.); 4,766,067 (Biswas); 4,656,134 (Ringold) and RNA-mediated amplification using antisense RNA to a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238, (Malek et al) with the trade name NASBA), the entire contents of those references are hereby incorporated by reference (see, for example, Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.); or J. Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New Yo rk, NY).

[0125]Conveniently, the method steps described herein, such as amplification, sequencing, and the like, may or may not be performed in a multiplex analysis format using a solid phase on which a plurality of substrates, e.g., antigens, and the like, can be immobilized, e.g. , in the form of a matrix (array). In some embodiments, such a matrix is a protein biochip. Using protein biochips, hundreds and even thousands of antigens can be tested. The term "matrix", "microchip" or "biochip" as used herein refers to a solid support having a generally flat surface to which an adsorbent is attached. Often the surface of a biochip contains many "address" positions, each of which has its own adsorbent. Biochips can be adapted and used as a probe interface and therefore function as probes. "Protein biochip" refers to a biochip adapted to recognize and capture polypeptides. Many protein biochips are described in this prior art. Methods for obtaining polypeptide matrices are described, for example, in De Wildt et al, 2000, Nat. Biotechnol. 18:989-994; Lueking et al., 1999, Anal. Biochem. 270:103-1 11; Ge, 2000, Nucleic Acids Res. 28, e3, 1- V_H; MacBeath and Schreiber, 2000, Science 289: 1760-1763; WO 01/40803 and WO 99/51773A1. The use of matrices allows multiple steps, such as sorting, to be performed in a automated and/or high-throughput manner. Template polypeptides can be detected at high speed, for example, using a commercially available robotic device, such as those manufactured by Genetic MicroSystems or BioRobotics. The matrix substrate can be, for example, nitrocellulose, plastic, glass, such as surface-modified glass. The matrix may also include a porous support substrate such as acrylamide, agarose, or other polymer.

[0126] When captured on a biochip, analyzed molecules can be detected by various methods selected from, for example, gas phase ion spectrometry, optical method, electrochemical method, atomic force microscopy method, and radio frequency method. Of particular interest is the use of mass spectrometry and, in particular, SELDI. Optical methods include, for example, detection (detection) of fluorescence, luminescence, chemiluminescence, optical density, reflectivity, transmittance, birefringence or refractive index (for example, using surface plasma resonance, ellipsometry, resonant mirror method, waveguide grating method or interferometry) . Optical techniques include microscopy (both confocal and non-confocal), imaging techniques, and non-imaging techniques. Immunoassays of various formats (eg, ELISA) are a popular method for detecting analytes (molecules of interest) fixed on a solid phase. Electrochemical methods include voltammetry and amperometry methods. Radio frequency methods include multipolar resonant spectroscopy.

[0127] In some embodiments of the invention, techniques are used that are designed to work with individual cells or where a selection of specific populations of cells is needed. One exemplary technique involves a special tool that can be used in FACS to guide individual cells into individual container cells. Such devices are commercially available and are well known in the art. Such devices are useful for packaging single cells into selected compartments, such as standard 96-well microtiter plates. Alternatively, cells can be delivered to a microtiter plate at an appropriate dilution to allow single cell deposition.

[0128] The second method is PCR performed on single immune cells to amplify the V _H and V _L or Vα and Vβ or Vγ and Vδ segments, in addition to the optional amplification of the single immune cell transcriptome. In some embodiments, single cell PCR is used to maintain native mating of V _L and V _H or Vα and Vβ or Vγ and Vδ in a single cell. The specificity of an antibody or TCR is determined by the complementarity determining regions (CDRs) in the V _L region and the V _H region, or the Vα and Vβ or Vγ and Vδ regions, respectively.

[0129] Single cell PCR methods are well known (eg, Larrick, JW et al., Bio/Technology 7: 934 (1989)). For example, antibody-producing B cells from a B cell library or TCR-producing TCR from a T cell library can be fixed with a fixative solution or a solution containing a chemical such as formaldehyde, glutaraldehyde, or the like. The cells are then permeabilized with a permeabilizing solution containing, for example, a detergent. The fixation and permeabilization process must provide sufficient porosity to allow the introduction of enzymes, nucleotides and other reagents into cells without excessive destruction of their cellular compartments or nucleic acids in them. The addition of enzymes and nucleotides can then be introduced into cells to allow reverse transcription of cellular mRNA, including V _H and V _L or Vα and Vβ or Vγ and Vδ mRNA, for example, into the appropriate cDNA sequences. In other examples, single cell PCR can be performed in a solution of lysed non-fixed cells, as described here.

[0130] Reverse transcription can be carried out in one step or optionally together with a PCR procedure using reverse transcriptase, a sufficient number of four dNTPs, and primers that bind to the mRNA, providing a 3'-hydroxyl group for the reverse transcriptase to initiate polymerization. Target-specific primers and/or random hexameric oligonucleotide primers can be used to initiate the reverse transcription reaction and generate high quality sequencing libraries.

[0131] For target sequences, any primer that is complementary to the target mRNA can be used, but it is preferable to use primers that are complementary to the 3' end of the V _H and V _L or Vα and Vβ or Vγ and Vδ molecules to facilitate mRNA variable region selection. . Numerous studies have shown that degenerate polynucleotides can be prepared as 5' primers for V _H and V _L or Vα and Vβ or Vγ and Vδ. The combinatorial library method for obtaining target molecules is based on the use of such primers. In addition, numerous experiments have shown that PCR can amplify gene segments of interest such as V _H and V _L or Vα and Vβ or Vγ and Vδ from a single cell. With the ability to work on even a single cell, this PCR technique allows the production of antibodies even when the immune cells of interest are encountered at low frequency.

[0132] In some embodiments, after FACS sorting, cells from the immune cell library are pooled and reverse transcription-PCR is performed on the entire cell pool. Generating mRNA for cloning antibodies or TCR targets is readily accomplished using well-known techniques for generating and characterizing antibodies or TCRs (see, for example, "Antibodies: A Laboratory Manual, 1988"; incorporated herein by reference). For example, total RNA is isolated from a B cell library using appropriate methods that are standard and accepted in the art. The cDNA is then synthesized from the RNA by appropriate methods, for example, using random hexamer polynucleotides, or C gene family or C gene family specific primers, or V gene family or V gene family specific primers. Again, these processes are known to those skilled in the art, as explained above. Libraries of nucleic acid molecules derived from B-cell or T-cell libraries, for example libraries of RNA or cDNA molecules derived from such B- or T-lymphocytes, can be cloned into expression vectors to form expression libraries. In some embodiments, only a V _H or Vα or Vγ domain derived from an immune cell library is amplified to create a library of V _H or Vα or Vγ domains. A V _L or Vβ or Vδ library from another source is used in combination with a V _H or Vα or Vγ library to generate antibodies or TCRs using the methods described here. Libraries of antibody fragments or TCRs can be constructed by combining V _H and V _L or Vα and Vβ or Vγ and Vδ libraries in any manner known to those skilled in the art. For example, each library can be created in different vectors and the vectors recombined in vitro or in vivo. Alternatively, the libraries can be cloned sequentially into the same vector, or brought together by PCR and then cloned. Assembly PCR can also be used to combine V _H and V _L or Vα and Vβ or Vγ and Vδ with DNA encoding a flexible peptide spacer to form single-stranded Fv (scFv) libraries, as described elsewhere herein. In yet another method, intracellular assembly by PCR is used to combine the V _H and V _L or Vα and Vβ or Vγ and Vδ genes in lymphocytes by PCR, and then the repertoire of related genes is cloned.

1. Target polynucleotides

[0133] In embodiments, the methods provided herein are directed to the amplification and sequencing of a target polynucleotide molecule, such as an intracellular polynucleotide molecule. In some instances, the methods provided herein are directed to the amplification and sequencing of two or more regions of a target polynucleotide molecule. In some cases, the methods provided herein are directed to the amplification and sequencing of two or more target polynucleotide molecules, such as two or more molecules in a natural pair. In one aspect, the target polynucleotides are RNA. In one aspect, the target polynucleotides are genomic nucleic acids. DNA derived from the genetic material of a particular organism's chromosomes may be genomic DNA.

[0134] In some embodiments, reference to "target nucleic acid molecule", "target polynucleotide", "target polynucleotide molecule" refers to any nucleic acid of interest.

[0135] In some embodiments, the target polynucleotides include sequences containing the variable regions of an immune receptor, such as an antibody or TCR, produced by an immune cell.

[0136] In some embodiments, the target polynucleotides comprise two or more immune receptor chains that are naturally paired to form an immunoreceptor or binding fragment thereof. In some embodiments, target polynucleotides include sequences containing the heavy chain variable region of an antibody produced by an immune cell. In some embodiments, target polynucleotides include sequences containing the light chain variable region of an antibody produced by an immune cell. In some embodiments, target polynucleotides include sequences containing the heavy chain variable region and sequences containing the light chain variable region of an antibody produced by the same immune cell.

[0137] Thus, the term "antibody" is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and their functional (antigen-binding) antibody fragments, including fragments of antigen-binding (Fab) fragments, F(ab ') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments including single chain variable fragments (scFv), and single domain antibody fragments (e.g., sdAb, sdFv, nanobody). The term encompasses engineered and/or otherwise modified forms of immunoglobulins such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, human-adapted and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, tribodies, and tetra bodies, di-scFv tandem, tri-scFv tandem. Unless otherwise indicated, the term "antibody" is to be understood to encompass functional fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0138] It is known in the art that the terms "complementarity determining region" and "CDR" are synonymous with the terms "hypervariable region" or "HVR" and refer to non-contiguous amino acid sequences in the variable regions of an antibody that confer specificity and/or specificity to an antigen. or binding affinity. Typically, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3) and three more CDRs are located in each light chain variable region (CDR-L1, CDR-L2 and CDR-L3). The terms "framework regions" and "FR/framework regions" are known in the art and refer to non-CDR portions of the heavy and light chain variable regions. Typically, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3 and FR-L4).

[0139] The exact boundaries of the amino acid sequence of a given CDR or FR can be easily determined using any of a number of well known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol . Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme), Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme), and Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme).

[0140] Thus, unless otherwise indicated, the terms "CDR" or "complementarity determining region", or individual specified CDRs (for example, "CDR-H1, CDR-H2) of a given antibody or fragment thereof, for example, a variable region, should be understood to encompass some (or specific) domain defining complementarity as defined by any of the aforementioned schemes. For example, if a particular CDR (eg, CDR-H3) is indicated to contain the amino acid sequence of the corresponding CDR in that V _H or V _L amino acid sequence, then that CDR is understood to have the sequence corresponding to the CDR (eg, CDR-H3) in the variable areas as defined by any of the above schemes. In some embodiments, specific CDR sequences are indicated.

[0141] Similarly, unless otherwise indicated, it is to be understood that the FR or a single specified FR (e.g., FR-H1, FR-H2) of a given antibody or region, e.g., variable region, encompasses some (or specific) framework region, as defined by any of the known schemes. In some cases, a scheme is specified to identify specific CDRs, FRs or FRs, or CDRs such as CDRs as determined by the Kabat, Chothia or Contact method. In other cases, the specific amino acid sequence of the CDR or FR is indicated.

[0142] The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (V _H and V _L , respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single V _H or V _L domain may be sufficient to confer antigen-binding specificity. that bind a particular antigen can be isolated using the V _H or V _L domain of an antigen-binding antibody to screen a library of complementary V _L or V _H domains, respectively.See, for example, Portolano et al., J. Immunol.150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0143] Among the provided antibodies are antibody fragments. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to: Fv, Fab, Fab', Fab'-SH, F(ab') 2; dimers; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

[0144] Immunoglobulins (Igs), expressed by B cells, in some aspects are proteins consisting of four polypeptide chains, two heavy chains (IgH) and two light chains (IgL), forming the H2L2 structure. Each pair of IgH and IgL chains contains a hypervariable domain consisting of a V _L region and a V _H region, and a constant domain. Chains of IgH Ig belong to several types: μ, δ, γ, α and β. The diversity of Igs in a particular subject (human) is mainly determined by the hypervariable domain. Like TCR, the V domain of the IgH chains is created by combinatorial joining of the V _H , DH and JH gene segments. The independent addition and deletion of nucleotides at the V _H -DH, DH-JH and V _H -JH borders during the Ig gene rearrangement further increases the diversity of hypervariable domain sequences. Here, immunocompetence is reflected in the diversity of Igs.

[0145] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (V _H and V _L , respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single V _H or V _L domain may be sufficient to confer antigen-binding specificity. that bind a particular antigen can be isolated using the V _H or V _L domain from an antigen-binding antibody to screen for a library of complementary V _L or V _H domains, respectively.See, for example, Portolano et al., J. Immunol.150 : 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0146] The term "hypervariable region" refers to the amino acid residues of an antibody or TCR that are responsible for antigen binding. The hypervariable region contains the amino acid residues of the complementarity determining region or CDR. Framework or FR residues are those variable domain residues that are not hypervariable region residues as defined herein.

[0147] Among the presented antibodies are also fragments of antibodies. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2; dimers; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

[0148] Single domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody.

[0149] Antibody fragments can be obtained by various methods, including, but not limited to: proteolytic cleavage of intact antibodies, as well as production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments containing structures that do not occur naturally, such as fragments with two or more antibody regions or chains connected by synthetic linkers, e.g., peptide linkers, and/or those that may not be produced by enzymatic cleavage of native intact antibodies. In some aspects, the antibody fragments are scFv.

[0150] Antigen-binding polypeptides also include heavy chain dimers such as, for example, camelid and shark antibodies. Camelid and shark antibodies contain a homodimeric pair of two chains of V-like and C-like domains (neither has a light chain). Because the V _H region of the camelid IgG heavy chain dimer should not cause hydrophobic interactions with the light chain, the heavy chain region that normally binds to the light chain is changed to hydrophilic amino acid residues in camelids. The V _H domains of heavy chain dimeric IgGs are referred to as VHH domains. Shark Ig-NARs contain a homodimer of one variable domain (called the V-NAR domain) and five C-like constant domains (C-NAR domains). In camelids, the diversity of the antibody repertoire is defined by CDRs 1, 2, and 3 in the V _H or VHH regions. The CDR3 in the camelid VHH region is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7 (9): 1129).

[0151] An antibody "human adapted" is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A human-adapted antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "human-adapted form" of a non-human antibody refers to a non-human variant of an antibody that has been altered/adapted, usually to reduce immunogenicity in humans while retaining the specificity and affinity of the non-human parent antibody. In some embodiments, certain FR residues in a human-adapted antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0152] Among the provided antibodies are human antibodies. A "human antibody" is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human cell or non-human source that uses the human antibody repertoire or other sequences encoding human antibodies, including human antibody libraries. The term excludes forms of antibodies that are not human but adapted for humans and contain "non-human" antigen-binding regions, such as those in which all or substantially all of the CDRs are not "human" (i.e., not human).

[0153] Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact human variable region antibodies in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci replacing endogenous immunoglobulin loci or present outside the chromosomes or randomly integrated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are usually inactivated. Human antibodies can also be obtained from human antibody libraries, including phage display libraries and cell-free libraries containing sequences encoding antibodies derived from the human repertoire.

[0154] Among the provided antibodies are monoclonal antibodies, including fragments of monoclonal antibodies. The term "monoclonal antibody" in the context of the present description refers to an antibody derived from a population or within a population of essentially homogeneous antibodies, that is, where the individual antibodies that make up the population are identical, except for the possible variants containing naturally occurring mutations or arising in during the production of a monoclonal antibody preparation, but such variants are usually present in small quantities. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope of the antigen. The term should not be construed as requiring the production of an antibody by any particular method. A monoclonal antibody can be obtained by various methods, including, but not limited to, such as: obtaining from hybridoma, recombinant DNA methods, phage display method, and other methods for obtaining antibodies.

[0155] In some embodiments, target polynucleotides include sequences containing the variable region of the TCR alpha chain produced by an immune cell. In some embodiments, the target polynucleotides include sequences containing the variable region of the beta chain of a TCR produced by an immune cell. In some embodiments, the target polynucleotides include sequences containing the alpha chain variable region of a TCR and sequences containing the beta chain variable region of a TCR produced by the same immune cell. In some embodiments, the target polynucleotides include sequences containing the variable region of the TCR gamma chain produced by an immune cell. In some embodiments, the target polynucleotides include sequences containing the delta chain variable region of a TCR produced by an immune cell. In some embodiments, target polynucleotides include sequences containing the variable region of the TCR gamma chain and sequences containing the variable region of the delta chain of a TCR produced by the same immune cell.

[0156] In some embodiments, a TCR encompasses full-length TCRs as well as their antigen-binding portions or antigen-binding fragments (also referred to as MHC-peptide-binding fragments). In some embodiments, the TCR is an intact or full length TCR. In some embodiments, the TCR is an antigen-binding portion that is smaller than the full-length TCR, but that binds to a specific antigenic peptide associated (i.e., in context) with an MHC molecule, i.e., an MHC-peptide complex. In some cases, the antigen-binding portion or a TCR fragment may contain only a subset of the structural domains of the full-length or intact TCR, but still be able to bind the epitope (eg, MHC-peptide complex) to which the full-length TCR binds. In some instances, the antigen-binding portion or fragment of the TCR contains TCR variable domains, such as the TCR variable α chain and the TCR variable β chain, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as where each chain contains three complementarity-determining regions. The term includes polypeptides or proteins having a binding domain that is an antigen-binding domain or homologous to an antigen-binding domain. The terms also cover grafted (engineered) antibodies carrying a complementarity determining region (CDR), TCR and other human-adapted antibodies and TCRs (including CDR modifications and framework region modifications). It should be noted that although only immunoglobulin chains (e.g., heavy chains and light chains) can be referred to, the disclosed invention can be applied to many other different types of paired sequences, for example, pairs of T cell receptor chains (TCRα and TCRβ chains and chains TCRγ and TCRδ) and are not limited to immunoglobulins.

[0157] The ability of T cells to recognize antigens associated with various cancers or infectious organisms is provided by their TCR, which consists of both alpha (α) chain and beta (β) chain or gamma (γ) and delta (δ ) chains. The proteins that make up these chains are encoded by DNA, which uses a unique mechanism to create a huge variety of TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides represented by MHC class I and II proteins on the surface of antigen presenting cells (APCs). Binding of a TCR to an antigenic peptide on the surface of an APC is a central event in T cell activation occurring at the immunological synapse at the point of contact between the T cell and the APC.

[0158] Each TCR contains variable complementarity determining regions (CDRs) and framework regions (FRs). Amino acid sequence loops of the third complementarity-determining region (CDR3) of the α- and β-chain variable domains largely determine T-cell αβ sequence variability resulting from recombination between the variable (Vβ), diverse (Dβ) and joining (Jβ) regions gene at the β-chain locus and between analogous segments of the Vα and Jα genes at the α-chain gene locus, respectively. The presence of many such gene segments at the α and β loci of the TCR chain allows for the coding of a large number of different CDR3 sequences. The independent addition and deletion of nucleotides at the Vβ-Dβ, Dβ-Jβ and Vα-Jα junctions during the TCR gene rearrangement process further increases the diversity of CDR3 sequences. In this regard, immunocompetence is expressed in the diversity of TCRs.

[0159] TCR fragments are also provided, including antigen-binding fragments. In some embodiments, the TCR is an antigen-binding portion thereof, such as a full-length TCR variant lacking its transmembrane and/or cytoplasmic region(s), which may be referred to as a fully soluble TCR. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single stranded TCR (scTCR) such as scTCR having the structure described in PCT Patent Publication Nos. WO 03/020763, WO 04/033685, or WO 2011/044186. In some embodiments, the TCR is a single chain TCR fragment containing an alpha chain variable region linked to a beta chain variable region, such as scTv. In some embodiments, scTv is also referred to as scFv.

[0160] A single chain Fv or scFv refers in some aspects to antibody or TCR fragments that include antibody variable heavy chain (V _H ) and light chain variable (V _L ) domains or variable alpha or gamma chain domains (Vα or Vγ) or TCR variable beta or delta chain (Vβ or Vδ) domains, where these domains are present in the same polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains, or the Vα and Vβ domains, or the Vγ and Vδ domains, which allows the scFv to form the desired structure for antigen binding.

[0161] Diatelo refers in some aspects to small fragments of an antibody and/or TCR with two antigen-binding sites, wherein these fragments contain a V _H linked to a V _L in the same polypeptide chain (V _H - V _L ), or a Vα linked with Vβ in the same polypeptide chain (Vα-Vβ) or Vγ linked to Vδ in the same polypeptide chain (Vγ-Vδ). Using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with complementary domains on the other strand and create two antigen-binding sites. Typical diabodies are described more fully, for example, in EP404097 and WO93111161.

[0162] A bispecific antibody or bispecific TCR, in some aspects, refers to an antibody or TCR that exhibits specificity for two different types of antigens. As used herein, the terms specifically include, without limitation, antibodies and TCRs that exhibit binding specificity for a target antigen and other target that facilitates delivery to a particular tissue. Similarly, multispecific antibodies and TCRs have two or more binding specificities.

[0163] A linear antibody or "linear TC" in some aspects refers to a pair of tandem Fd segments (eg, V _H -CH1 - V _H -CH1 or Vα-Cα1-Vα-Cα1) that form a pair of antigen-binding regions. Linear antibodies and TCRs can be bispecific or monospecific, for example as described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995).

[0164] An antigen-binding domain, in some aspects, refers to one or more antibody or TCR fragments that retain the ability to specifically bind to an antigen. Non-limiting examples of antibody fragments included in such terms include: (i) a Fab fragment, a monovalent fragment consisting of V _L , V _H , CL and CH1 domains; (ii) fragment F (ab ') 2, a divalent fragment containing two Fab fragment connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V _H and CH1 domains; (iv) an Fv fragment containing the V _L and V _H domains of one antibody arm, including scFvs, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544,546) that contains a V _H domain; and (vi) but are not limited to a dedicated CDR. Also included in this definition are antibodies containing one heavy chain and one light chain and TCRs with one alpha chain or one beta chain.

[0165] "F(ab') 2" and "Fab'" fragments can be obtained by treatment of Ig with a protease such as pepsin and papain and include antibody fragments obtained by cleavage of an immunoglobulin near the disulfide bonds that exist between the hinge regions in each of two heavy chains. For example, papain cleaves IgG before the disulfide bonds that exist between the hinge regions in each of the two heavy chains to form two homologous antibody fragments in which the light chain consists of V _L and CL and the heavy chain fragment consists of V _H and CHγ1 (region γ1 in the heavy chain constant region) linked at their C-terminal regions via a disulfide bond. Each of these two homologous antibody fragments is called a " Fab ". Pepsin also cleaves IgG downstream of the disulfide bonds that exist between the hinge regions in each of the two heavy chains to form a slightly larger antibody fragment than the fragment in which the two aforementioned ' Fab 's are linked at the hinge region. This antibody fragment is referred to as F('ab') 2 . The Fab fragment also contains a light chain constant domain and a heavy chain first constant domain (CH1). ' Fab ' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is here the designation of Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') 2 antibody fragments are initially prepared as pairs of Fab' fragments that have hinged cysteines in between.

[0166] Fv in some aspects refers to an antibody or TCR fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable region and one light chain variable domain, or one TCRα chain and one TCRβ chain, or one TCRγ chain and one TCRδ chain in tight non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to form an antigen-binding site on the surface of the V _H -V _L dimer or the Vα-Vβ dimer or the Vγ-Vδ dimer. Collectively, the combination of one or more CDRs from each of the V _H and V _L chains, or the Vα and Vβ chains, or the Vγ and Vδ chains, confers antigen-binding specificity to the antibody or TCR. For example, it should be understood that, for example, CDRH3 and CDRL3 may be sufficient to confer antigen-binding specificity on an antibody or TCR when transferred into V _H and V _L chains or Vα and Vβ chains or Vγ and Vδ chains in a selected recipient antibody, TCR or antigen-binding agent thereof. fragment, and this combination of CDRs can be tested for binding, affinity, etc. Even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind an antigen, albeit probably at a lower affinity than in combination with the second variable domain. In addition, although the two domains of the Fv fragment (V _L and V _H or Vα and Vβ or Vγ and Vδ) are encoded by separate genes, they can be combined by recombinant methods using a synthetic linker that allows them to exist as a single-stranded protein chain in which the V _L and V _H or Vα and Vβ or Vγ or Vδ regions join to form monovalent molecules (known as single chain Fv (scFv); Bird et al. (1988) Science 242: 423-426; Huston et al. et al. ( 1988) Proc Natl Acad Sci USA 85: 5879-5883 and Osbourn et al (1998) Nat Biotechnol 16: 778). Such scFvs are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Any V _H and V L sequences of a particular scFv can be linked to the Fc region of the cDNA or genomic sequences to create expression vectors encoding full-length Ig molecules (eg, IgG) or other isotypes. V _H and V _L can also be used to generate Fab, Fv or other Igs fragments using either protein chemistry technology or recombinant DNA technology.

[0167] The term "germline sequence" refers to the genetic sequence from the germline (haploid gametes and those diploid cells from which they are formed). The germline DNA contains a plurality of gene segments that encode one Ig heavy or light chain, or one TCRα or TCRβ chain, or one TCRγ or TCRδ chain. These gene segments are carried in germ cells but cannot be transcribed and translated until they are grouped into functional genes. During the differentiation of B cells and T cells in the bone marrow, these gene segments are randomly intermixed by a dynamic genetic system capable of generating more than 108 specific combinations. Most of these gene segments have been published and compiled by the germline database.

[0168] In some embodiments, the implementation of the immune molecule may be or may be a neutralizing antibody or a neutralizing TCR. In some aspects, a neutralizing antibody or TCR is an antibody or TCR that inhibits the replication of a pathogen, such as a virus or bacterium, regardless of the mechanism by which neutralization is achieved.

[0169] In some embodiments, a sample, such as a population of cells or a single cell, may contain an immune repertoire, such as an antibody repertoire or a TCR repertoire, and such a repertoire may be determined using the provided methods. In some embodiments, an antibody repertoire or TCR repertoire refers to a collection of antibodies, TCRs, or fragments thereof. In some embodiments, an antibody repertoire may, for example, be used to select a particular antibody or screen for a particular property, such as binding capacity, binding specificity, gastrointestinal transport capacity, stability, affinity, and the like. This term specifically includes antibody and TCR libraries, including all forms of combinatorial libraries, such as, for example, antibody phage display libraries, including, but not limited to, Fv single chain (scFv) libraries and Fab antibody phage display libraries from any source, including naive, synthetic and semi-synthetic libraries.

[0170] Target polynucleotides can be obtained from almost any source, and using methods known in this field. For example, target polynucleotides can be directly isolated without amplification using methods known in the art, including, without limitation, isolating genomic DNA or mRNA fragments from an organism or cell (eg, an immune cell) to obtain target polynucleotides. The term "target polynucleotide" may also include cDNA generated from RNA (such as mRNA) by reverse transcription-PCR. In some cases, the target polynucleotide is an RNA molecule. In some instances, the target polynucleotide is an mRNA or cDNA molecule derived from an mRNA molecule. In some cases, the target polynucleotide is an mRNA molecule or a cDNA molecule derived from an mRNA molecule from a single immune cell. In some cases, target polynucleotides are mRNA molecules or cDNA molecules derived from mRNA molecules from individual immune cells. In some cases, the target polynucleotides are mRNA molecules encoding an antibody sequence from a single immune cell. In some instances, target polynucleotides are mRNA molecules encoding antibody heavy chain sequences from individual immune cells. In some instances, target polynucleotides are mRNA molecules encoding an antibody heavy chain sequence from a single immune cell. In some instances, target polynucleotides are mRNA molecules encoding antibody light chain sequences from individual immune cells. In some instances, target polynucleotides are mRNA molecules encoding an antibody light chain sequence from a single immune cell. In some instances, target polynucleotides are mRNA molecules encoding antibody variable sequences from individual immune cells. In some instances, target polynucleotides are mRNA molecules encoding an antibody variable sequence from a single immune cell. In some instances, target polynucleotides are mRNA molecules encoding antibody light chain variable sequences from individual immune cells. In some instances, target polynucleotides are mRNA molecules encoding a light chain variable sequence of an antibody from a single immune cell. In some instances, target polynucleotides are mRNA molecules encoding antibody heavy chain variable sequences from individual immune cells. In some instances, the target polynucleotides are mRNA molecules encoding an antibody heavy chain variable sequence from an immune cell. In some cases, the target polynucleotide may be a nucleic acid cell-free fraction, such as DNA or RNA. In some instances, the target polynucleotides are mRNA molecules encoding alpha, beta, gamma, and/or delta TCR chain variable sequences from individual immune cells.

[0171] The methods described herein can be used to create a library of polynucleotides from one or more sequencing target polynucleotides. Target polynucleotides include any polynucleotides of interest that are not products of an amplification reaction. For example, a target polynucleotide may include a polynucleotide in a biological sample. For example, target polynucleotides do not include PCR reaction products. For example, target polynucleotides may include the polynucleotide template used to generate the amplification reaction products, but do not include the amplification products themselves. For example, target polynucleotides may include the polynucleotide template used to generate the products of the reverse transcription reaction or primer elongation reaction, as well as the products of the reverse transcription reaction or primer elongation reaction. For example, target polynucleotides include polynucleotides of interest that can be subjected to a reverse transcription reaction or a primer elongation reaction. For example, target polynucleotides include RNA or DNA. For example, target polynucleotides include cDNA. In some embodiments, the target RNA polynucleotides are mRNA. In some embodiments, the target RNA polynucleotides are polyadenylated. In some embodiments, the RNA polynucleotides are not polyadenylated. In some embodiments, the target polynucleotides are DNA polynucleotides. DNA polynucleotides can be represented by genomic DNA. DNA polynucleotides may contain exons, introns, untranslated regions, or any combination thereof.

[0172] In some embodiments, libraries can be derived from two or more regions of a target polynucleotide. In some embodiments of the methods, libraries can be generated from two or more target polynucleotides. In some embodiments, target polynucleotides are genomic nucleic acids or DNA derived from chromosomes. In some embodiments, target polynucleotides include sequences containing a variant, such as a polymorphism or mutation. In some embodiments, target polynucleotides include DNA and not RNA. In some embodiments, target polynucleotides include RNA and not DNA. In some embodiments, target polynucleotides include DNA and RNA. In some embodiments, the target polynucleotide is an mRNA molecule. In some embodiments, the target polynucleotide is a DNA molecule. In some embodiments, the target polynucleotide is a single stranded polynucleotide. In some embodiments, the target polynucleotide is a double-stranded polynucleotide. In some embodiments, the target polynucleotide is a single stranded double stranded polynucleotide.

[0173] Target polynucleotides can be obtained from any biological sample using methods known in the art. In some embodiments, target polynucleotides are directly isolated without amplification. Direct isolation methods are known in the art. Non-limiting examples include the extraction of genomic DNA or mRNA from a biological sample, organism, or cell.

[0174] In some embodiments, one or more target polynucleotides are purified from a biological sample. In some embodiments, the target polynucleotide is not purified from the biological sample in which it is contained. In some embodiments, the target polynucleotide is isolated from a biological sample. In some embodiments, the target polynucleotide is not isolated from the biological sample in which it is contained. In some embodiments, the target polynucleotide may be a cell-free fraction nucleic acid. In some embodiments, the target polynucleotide may be a fragmented nucleic acid. In some embodiments, the target polynucleotide may be a transcribed nucleic acid. In some embodiments, the target polynucleotide is a modified polynucleotide. In some embodiments, the target polynucleotide is an unmodified polynucleotide.

[0175] In some embodiments, the target polynucleotide is a polynucleotide from a single cell. In some embodiments, the target polynucleotides are derived from single cells. In some embodiments, the target polynucleotide is a polynucleotide from a sample containing a plurality of cells.

[0176] In some embodiments, the target polynucleotide encodes a biomarker sequence. In some embodiments, the target polynucleotide encodes two or more biomarker sequences. In some embodiments, a plurality of target polynucleotides encode a biomarker sequence. In some embodiments, a plurality of target polynucleotides encode two or more biomarker sequences. In some embodiments, a plurality of target polynucleotides encode 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more biomarker sequences.

[0177] In some embodiments, the plurality of target polynucleotides comprise a panel of immunoglobulin sequences. In some embodiments, the plurality of target polynucleotides comprise a panel of TCR sequences. For example, a panel of immunoglobulin sequences may be V _H and/or V _L sequences. In some embodiments, the immunoglobulin or TCR sequence panel contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunoglobulin or TCR sequences. In some embodiments, an immunoglobulin or TCR sequence panel contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 3000, 4000 5000 6000, 7000, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 8000, 9000, 10,000, 11000, 12000, 13000, 14000, 15000, 16000, 17000 18000, 19000, 20,000, 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, L × L0 ⁶ , 2×l0 ⁶ , 3×l0 6 , 4×l0 ⁶ , 5× ^{l0 6 ,} 6×l0 ⁶ , 7×l0 ⁶ , 8×l0 6 , 9×l0 ⁶ , l× ^{l0 7 ,} 2×l0 ⁷ , 3×l0 ⁷ , 4×l0 ⁷ , 5×l0 ⁷ , 6×l0 ⁷ , 7×l0 ⁷ , 8×l0 ⁷ , 9×l0 ⁷ , l×l0 8 , 2× ^{l0 8 ,} 3×l0 ⁸ , 4×l0 ⁸ , 5×l0 ⁸ , 6×l0 ⁸ , 7×l0 ⁸ , 8×l0 ⁸ , 9×l0 ⁸ , l×l0 9 , 2×l0 ⁹ , 3× ^{l0 9 ,} 4×l0 ⁹ , 5×l0 ⁹ , 6×l0 ⁹ , 7×l0 ⁹ , 8×l0 ⁹ , 9×l0 ⁹ , l×l0 ¹⁰ , 2×l0 ¹⁰ , 3×l0 ¹⁰ , 4×l0 ¹⁰ , 5×l0 ¹⁰ , 6×l0 ¹⁰ , 7×l0 ¹⁰ , 8×l0 ¹⁰ , 9×l0 ¹⁰ , l×l0 ¹¹ , 2×l0 ¹¹ , 3×l0 ¹¹ , 4×l0 ¹¹ , 5×l0 ¹¹ ,6×l0 ¹¹ , 7 ×l0 ¹¹ , 8×l0 ¹¹ , 9×l0 ¹¹ , l×l0 ¹² , 2×l0 ¹² , 3×l0 ¹² , 4×l0 ¹² , 5×l0 ¹² , 6×l0 ¹² , 7×l0 ¹² , 8 ×l0 ¹² , or 9×l0 ¹² immunoglobulin or TCR sequences. In some embodiments, the immunoglobulin or TCR sequence panel contains at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000 18000, 19000, 20,000, 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, L × L0 ⁶ , 2, 2 ×l0 ⁶ , 3×l0 6 , 4×l0 ⁶ , 5× ^{l0 6 ,} 6×l0 ⁶ , 7×l0 ⁶ , 8×l0 6 , 9×l0 ⁶ , l× ^{l0 7 ,} 2×l0 ⁷ , 3 ×l0 ⁷ , 4×l0 ⁷ , 5×l0 ⁷ , 6×l0 ⁷ , 7×l0 ⁷ , 8×l0 ⁷ , 9×l0 ⁷ , l×l0 8 , 2× ^{l0 8} , 3×l0 ⁸ , 4 ×l0 ⁸ , 5×l0 ⁸ , 6×l0 ⁸ , 7×l0 ⁸ , 8×l0 ⁸ , 9×l0 ⁸ , l×l0 9 , 2×l0 ⁹ , 3×l0 ⁹ , 4× ^{l0 9 ,} 5 ×l0 ⁹ , 6×l0 ⁹ , 7×l0 ⁹ , 8×, 9×l0 ⁹ , l×l0 ¹⁰ , 2×l0 ¹⁰ , 3×l0 ¹⁰ , 4×l0 ¹⁰ , 5×l0 ¹⁰ , 6×l0 ¹⁰ , 7×l0 ¹⁰ , 8×l0 ¹⁰ , 9×l0 ¹⁰ , l×l0 ¹¹ , 2×l0 ¹¹ , 3×l0 ¹¹ , 4×l0 ¹¹ , 5×l0 ¹¹ , 6×l0 ¹¹ , 7×l0 ^11.8 ×l 0 ¹¹ , 9×l0 ¹¹ , l×l0 ¹² , 2×l0 ¹² , 3×l0 ¹² , 4×l0 ¹² , 5×l0 ¹² , 6×l0 ¹² , 7×l0 ¹² , 8×l0 ¹² , or 9 ×l0 ¹² immunoglobulin or TCR sequences. In some embodiments, the immunoglobulin or TCR sequence panel contains about 10-20, 10-30, 10-40, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90 , 10-100, 50-60, 50-70, 50-80, 50-90, 50-100, 100-200, 100-300, 100-400, 100-300, 100-400, 100-500, 100 -600, 100-700, 100-800, 100-900, 100-1000, 500-600, 500-700, 500-800, 500-900, 500-1000, 1000-2000, 1000-3000, 1000-4000 1000-3000 1000-4000 1000-5000 1000-6000 1000-7000 1000-8000 1000-9000 1000-10000 5000-6000 5000-7000 -10000, 1-1×10 ⁵ , 1-2×10 ⁵ , 1-3×10 ⁵ , 1-4×10 ⁵ , 1-5×10 ⁵ , 1-6×10 ⁵ , 1-7×10 ⁵ , 1-8×10 ⁵ , 9×10 ⁵ , 1-1×10 ⁶ , 1-2×10 ⁶ , 1-3×10 ⁶ , 1-4×10 ⁶ , 1-5×10 ⁶ , 1 -6×10 ⁶ , 1-7×10 ⁶ , 1-8×10 ⁶ , 9×10 ⁶ , 1-l×10 ⁷ , 1-2×10 ⁷ , 1-3×10 ⁷ , 1-4× 10 ⁷ , 1-5×10 ⁷ , 1-6×10 ⁷ , 1-7×10 ⁷ , 1-8×10 ⁷ , 1-9×10 ⁷ , 1-1×10 ⁸ , 1-2×10 ⁸ , 1-3×10 ⁸ , 1-4×10 ⁸ , 1-5×10 ⁸ , 1-6×10 ⁸ , 1-7×10 ⁸ , 1-8×10 ⁸ , 1-9×10 ⁸ , 1-1×10 ⁹ , 1-2×10 ⁹ , 1-3×10 ⁹ , 1-4×10 ⁹ , 1-5×10 ⁹ , 1-6×10 ⁹ ,1-7×10 ⁹ , 1-8×10 ⁹ , 1-9×10 ⁹ , 1-1×10 ¹⁰ , 1-2×10 ¹⁰ , 1-3×10 ¹⁰ , 1-4×10 ¹⁰ , 1-5×10 ¹⁰ , 1-6×10 ¹⁰ , 1-7×10 ¹⁰ , 1-8×10 ¹⁰ , 1-9×10 ¹⁰ , 1-1×10 ¹¹ , 1-2×10 ¹¹ , 1 -3×10 ¹¹ , 1-4×10 ¹¹ , 1-5×10 ¹¹ , 1-6×10 ¹¹ , 1-7×10 ¹¹ , 1-8×10 ¹¹ , 1-9×10 ¹¹ , 1- 1×10 ¹² , 1-2×10 ¹² , 1-3×10 ¹² , 1-4×10 ¹² , 1-5×10 ¹² , 1-6×10 ¹² , 1-7×10 ¹² , 1-8 ×10 ¹² , or 1-9×10 ¹² immunoglobulin or TCR sequences.

[0178] In some embodiments, the target polynucleotide is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000 18,000, 19,000, or 20,000 bases or base pairs long. In some embodiments, the target polynucleotide is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000 , 19,000 or 20,000 bases or base pairs long. In some embodiments, the target polynucleotide is at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 18000 19,000 or 20,000 bases or base pairs long. In some embodiments, the target polynucleotide is about 10-20, 10-30, 10-40, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10 -100, 50-60, 50-70, 50-80, 50-90, 50-100, 100-200, 100-300, 100-400, 100-300, 100-400, 100-500, 100-600 100-700 100-800 100-900 100-1000 500-600 500-700 500-800 500-900 500-1000 1000-2000 1000-3000 -3000, 1000-4000, 1000-5000, 1000-6000, 1000-7000, 1000-8000, 1000-9000, 1000-10000, 5000-6000, 5000-7000, 5000-8000, 5000-900 bases or base pairs long. In some embodiments, the average length of target polynucleotides or fragments thereof may be less than about 100, 200, 300, 400, 500, or 800 bp, or less than about 5, 10, 20, 30, 40, 50, 60, 70, 80 less than about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100 kilobases. In some embodiments, the target sequence from a relatively short template, such as a sample containing the target polynucleotide, is about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bases. In some embodiments, sequencing data is matched to known or expected sequences using a database containing those sequences or immunoglobulin or TCR sequences associated with a disease or condition.

2. Collections of polynucleotides, such as transcriptomes

[0179] A collection of polynucleotides corresponding to genomic or transcriptome polynucleotides can be obtained from virtually any source, such as a single cell or multiple cells, and can be obtained using methods known in the art. For example, a collection of polynucleotides can be directly isolated from a single cell or multiple cells without amplification using methods known in the art, including, but not limited to, isolating a fragment of genomic DNA or mRNA from an organism or cell (e.g., from an immune cell) to obtain collections of polynucleotides. The collection of genomic or transcriptome polynucleotides may also include cDNA generated from RNA (such as mRNA) by reverse transcription-PCR. In some cases, the collection of polynucleotides is a collection of RNA molecules. In some cases, the collection of polynucleotides is a collection of mRNA molecules or a collection of cDNA molecules derived from mRNA molecules. In some cases, a collection of polynucleotides is a collection of mRNA molecules or cDNA molecules derived from mRNA molecules from a single immune cell. In some cases, a set of polynucleotides is a set of mRNA molecules or cDNA molecules derived from mRNA molecules from individual immune cells.

[0180] The methods described herein can be used to create a library containing a collection of polynucleotides from one or more cells for sequencing. The collection of polynucleotides can be derived from genomic DNA or RNA, such as mRNA transcripts from one or more cells of a biological sample. For example, genomic DNA or cellular RNA, such as mRNA, can be used as a template for generating products of an amplification reaction, such as a reverse transcription reaction or a primer elongation/extension reaction. In some examples, a collection of polypeptides may be derived from cDNA. In some embodiments, the collection of polynucleotides is generated from RNA, the polynucleotides are mRNA, and the collection is essentially the transcriptome of one or more cells of a biological sample. In some embodiments, the collection of polynucleotides is generated from RNA polynucleotides that are polyadenylated. In some embodiments, the RNA polynucleotides are not polyadenylated. In some embodiments, the collection of polynucleotides is generated from DNA polynucleotides. The DNA polynucleotides may be genomic DNA. DNA polynucleotides may contain exons, introns, untranslated regions, or any combination thereof. For example, a collection of polynucleotides of the present invention may contain genomic or transcriptome information of at least 5, 10, 100, 250, 500, 750, 1000, 2500, 5000, 10000, 25000, 50000, 75000, 10000, 250000 500000, 750000. 1,000,000, 2,500,000, 5,000,000, 7,500,000 or 10,000,000 carrier cells or individual cells such as groups or individual immune cells expressing various antibodies or TCRs.

[0181] In some embodiments, a collection of polynucleotides can be generated by a reverse transcriptase reaction or primer extension using random hexamer primers. In some embodiments, a collection of polynucleotides can be generated by a reverse transcriptase or primer extension reaction using a primer directed against the polyA nucleotide sequence. In some examples, a collection of polynucleotides can be created using reverse transcriptase or a primer extension reaction using oligo-dT. In some examples, the primers may be biotinylated. Collections of polynucleotides may optionally be purified after reverse transcription or primer extension reactions. For example, collections of polynucleotides generated using biotinylated primers can optionally be purified using streptavidin treatment methods. In other embodiments, the polynucleotides can be purified by one or more affinity purification methods, agarose gel electrophoresis.

3. Drip Libraries

[0182] In general, a drip library consists of several library elements combined into one collection. Libraries can vary in complexity from a single library element to lxl0 ¹⁵ or more library elements. Each element of the library represents one or more given components with a fixed concentration. The element may be, but is not limited to, cells, granules, amino acids, proteins, polypeptides, nucleic acids, polynucleotides, or small molecule chemical compounds. The element may contain an identifier such as a molecular barcode, a carrier barcode, or both.

[0183] A cell library element may include, but is not limited to, hybridomas, B cells, T cells, primary cells, cultured cell lines, cancer cells, stem cells, or any other cell type. Cell library elements are prepared by encapsulating a range of cells from one to tens of thousands into individual droplets. The number of encapsulated cells is usually determined by Poisson statistics based on cell density and droplet volume. However, in some cases, this number deviates from the Poisson statistic, as described in Edd et al., “Controlled encapsulation of single-cells into monodisperse picoliter drops.” Lab Chip, 8(8): 1262-1264, 2008. Nature's Cells Program allows libraries to be created in a streaming fashion, with many cell variants such as immune cells producing one antibody or TCR each, all present in one source medium, and then this the medium is broken up into individual carriers, such as drops or capsules, which contain at most one cell. Cells within individual carriers, such as drops or capsules, are then lysed and polynucleotides, such as cellular mRNA and genomic DNA, including target mRNA or DNA (e.g., heavy chain and light chain polynucleotides, and/or alpha and beta polynucleotides) are released from the carrier. chains and/or polynucleotides of gamma and delta chains) from lysed cells, which are barcoded with molecular and carrier barcodes and amplified. The dual barcode polynucleotide products are then combined or combined to create a library consisting of transcriptome or genomic and target library elements (e.g. heavy and light chain and/or alpha and beta chains and/or gamma and delta chains) library items. In particular, transcriptome and target libraries are combined.

[0184] A bead-based library element contains one or more beads and may also contain other reagents such as antibodies, enzymes, or other proteins. In the case where all elements of the library contain different types of granules, but are in the same environment, all elements of the library can be prepared from one source liquid or from multiple source liquids. In the case of cell libraries prepared in bulk from a set of variants, the library elements will be prepared from different source liquids. It is desirable to have only one cell per drop containing only a few drops containing more than one cell when starting with many cells. In some cases, deviations from the Poisson distribution can be obtained to provide improved droplet loading such that most droplets contain exactly one cell per drop, with a few exceptions for empty droplets or droplets containing more than one droplet.

[0185] In some embodiments, it is desirable to have exactly one carrier barcode polynucleotide per drop, and only a few droplets containing more than one carrier barcode polynucleotide when starting with multiple carrier barcode polynucleotides. In some cases, deviations from a Poisson distribution can be achieved to provide improved droplet loading such that most droplets contain exactly one carrier-barcoded polynucleotide per drop, with a few exceptions of empty droplets or drops containing more than one carrier-barcoded polynucleotide.

[0186] Examples of droplet libraries are collections of droplets having different contents ranging from beads, cells, small molecules, DNA, primers, antibodies, and barcoded polynucleotides. The droplet size varies from about 0.5 µm to 500 µm in diameter, corresponding to about 1 picoliter per 1 nanoliter. However, droplets can range in size from 5 µm to 500 µm. Preferably, the droplets have a diameter of less than 100 µm, from about 1 µm to 100 µm. The most preferred size is about 20 to 40 microns in diameter (10 to 100 picoliters). The preferred properties of droplet libraries under study include osmotic pressure balance, uniform size, and size ranges.

[0187] The droplets contained in the droplet library provided by the present invention preferably have a uniform size. That is, the diameter of any drop in the library will vary by less than 5%, 4%, 3%, 2%, 1%, or 0.5% compared to the diameter of other droplets in the same library. Uniform droplet size in a library can be critical to maintaining the stability and integrity of the droplets, and can also be important for the subsequent use of the library droplets for the various biological and chemical assays described herein.

[0188] The invention provides a droplet library containing a plurality of water droplets in an immiscible liquid, each droplet being preferably substantially uniform in size and containing a different library element. The invention provides a method for forming a drop library, comprising providing a single aqueous liquid medium containing various elements of the library, each element of the library being enclosed in an aqueous drop in an immiscible liquid.

[0189] In some embodiments, different types of elements (eg, cells or granules) are combined in a single source contained in the same medium. After the initial pooling, the elements are encapsulated into droplets to form a droplet library, in which each droplet with a different type of bead or cage represents a separate element of the library. Dilution of the stock solution ensures the encapsulation process. In some embodiments, the resulting drops will either contain one element or will contain nothing, that is, they will be empty. In other embodiments, the droplets formed will contain multiple copies of the library element. Encapsulated elements are typically options. In one example, the elements are immune cells of a blood sample and each immune cell is encapsulated to amplify and barcode nucleotide sequences of antibodies in the immune cells.

[0190] For example, in one type of emulsion library, there are library elements that have different particles, that is, cells or barcoded polynucleotides in a different medium that have been encapsulated before being combined. In one example, the specified number of library elements, that is, n is the number of different cells or barcoded polynucleotides, contained in different environments. Each of the library elements is individually emulsified and pooled, and at some point in time, each of the n-different combined library elements is combined and pooled. The resulting pool contains a plurality of water-in-oil emulsion droplets, each containing a different type of particle.

[0191] In some embodiments, the formed droplets will either contain one element of the library, or will not contain anything, that is, they will be empty. In other embodiments, the droplets formed will contain multiple copies of the library element. The content of the granules corresponds to the Poisson distribution, where there is a discrete probability distribution expressing the probability of a series of events occurring in a certain period of time, if these events occur at a known average rate and regardless of the time since the last event. The oils and surfactants used in the creation of the libraries prevent the exchange of the contents of the library between drops.

B. Methods for Obtaining a Dual Barcode Single Cell Polynucleotide Library

[0192] In some embodiments, methods for obtaining a polynucleotide library are provided, comprising the following steps: (a) lysing cells in each of a plurality of carriers, where each of said carriers contains a cell from a sample containing a population of cells, a plurality of oligonucleotides with a molecular barcode, and a first adapter containing an oligonucleotide with a carrier barcode; (b) producing in each carrier a plurality of single-stranded polynucleotides comprising (i) one or more single-stranded target polynucleotides complementary to one or more target polynucleotides present in the cell; and (ii) a collection of single-stranded polynucleotides, each of which is complementary to a polynucleotide in the cell; (c) attaching to each single stranded polynucleotide one of a plurality of oligonucleotides with a molecular barcode, resulting in a plurality of barcoded single stranded polynucleotides, each containing a unique molecular barcode; (d) attaching a first adapter containing the carrier-barcoded oligonucleotide or amplified product thereof to each of the barcoded single-stranded polynucleotides, resulting in a plurality of double-barcoded single-stranded polynucleotides, each of the double-barcoded single-stranded polynucleotides -code in one media contain the same media barcode; and (e) adding a second adapter to each of the double-barcoded single-stranded polynucleotides, wherein the first adapter and the second adapter are present at opposite ends in or near each of the double-barcoded single-stranded polynucleotides. Typical carriers (carrier containers) from which a polynucleotide library is built include wells, emulsions, drops, or microcapsules.

1. Media preparation

[0193] Any biological sample, including a sample containing a population of cells and containing polynucleotides, can be used in the methods described herein. Any sample containing a cell can generally be used in the methods described herein. For example, the sample may be a biological sample from the subject or obtained from such a sample containing RNA or DNA. The polynucleotides may be extracted from the biological sample, or the sample may be directly processed by methods without isolation or purification of the polynucleotides. The sample may be extracted or isolated DNA or RNA. The sample can also be total RNA or DNA isolated from a biological sample, or a cDNA library, viral or genomic DNA. In one embodiment, the polynucleotides are isolated from a biological sample containing a variety of other components such as proteins, lipids, and non-matrix nucleic acids. The nucleic acid template molecules can be derived from any cellular material derived from an animal, plant, bacterium, fungus, or any other cellular organism.

[0194] In certain embodiments, the polynucleotides are derived from a single cell, such as a cell present in a population of cells. Polynucleotides can be obtained directly from an organism or from a biological sample obtained from an organism. Any sample of tissue or body fluid can be used as a source of nucleic acid for use in the invention. Polynucleotides can also be isolated from cultured cells, such as a primary cell culture or cell line. In some embodiments, such a cell may be a blood cell, an immune cell, a tissue cell, or a tumor cell. In some embodiments, the cell is an immune cell, such as a B cell or a T cell. The B cell may be a plasmablast, a memory B cell, or a plasma cell. The cells or tissues from which the template nucleic acids are derived may be infected with a virus or other intracellular pathogen.

[0195] In some embodiments, a population of cells, such as a population containing immune cells, can be isolated from blood or other biological samples of a subject or host, such as a human or other animal, that has been immunized or is suffering from an infection, cancer, autoimmune disease or any other disease. In some embodiments, the person may or may not be diagnosed with a disease, and the person may or may not show symptoms of the disease. In some embodiments, the subject or host, such as a human, may be one that has been exposed to and/or one that can produce TCR against an infectious agent (e.g., viruses, bacteria, parasites, prions, etc.), antigen, disease or an antigen associated with a disease or condition, such as, for example, a tumor antigen. In some embodiments, immune cells can be derived from any biological sample containing T cells, such as those present in peripheral blood mononuclear cells (PBMCs), the spleen, or other lymphoid organ. In some embodiments, the immune cells are derived from a normal healthy subject's T cell source. In some embodiments, the immune cells are derived from a source of T cells from a patient subject. In some embodiments, CD4+ or CD8+ cells can be isolated or obtained. In some cases, peripheral blood mononuclear cells (PBMC) can be isolated or obtained. In some cases, tumor infiltrating lymphocytes (TILs) can be isolated or obtained.

[0196] In certain embodiments, the antibody or TCR-producing immune cells can be isolated from blood or other biological samples of a subject or host, such as a human or other animal, that has been immunized or is suffering from an infection, cancer, an autoimmune condition, or any other diseases, to identify pathogenic, neoplastic and/or disease-specific antibodies or TCRs that may be of clinical significance. For example, a person may/has not been diagnosed with a disease, may or may not have symptoms of the disease. For example, such a person may be someone who has been exposed to and/or who can produce TCR against an infectious agent (eg, viruses, bacteria, parasites, prions, etc.), an antigen, or a disease. In some examples, the animal may be one that has been exposed to and/or that can produce beneficial antibodies or TCRs against an infectious agent (eg, viruses, bacteria, parasites, prions, etc.), antigen, or disease. In some examples, an animal, such as a human, no longer exhibits symptoms of the disease or condition. Certain immune cells from immunized hosts produce antibodies or TCRs to one or more target antigens and/or one or more unknown antigens. In the present invention, the pool of lymphocytes can be enriched with the desired immune cells by any suitable method, such as screening and cell sorting using fluorescence activated cell sorting (FACS), magnetically activated cell sorting (MACS), panning, or other screening method to obtain a plurality of immune cells from a sample, such as an immune cell library, before antibody chains are sequenced, antibodies generated, or expression library(s) generated. Unlike prior art enrichment methods that could provide only a few subsets of immune cells expressing different antibodies and therefore only a few naturally occurring combinations of variable domains, the immune cell library of the present invention contains at least two subsets of immune cells or individual cells expressing various antibodies or TCRs. For example, an immune cell library of the present invention may contain at least 5, 10, 100, 250, 500, 750, 1000, 2500, 5000, 10000, 25000, 50000, 75000, 10000, 250000, 500000, 750000, 100000 2500000, 5000000, 7500000 or 10000000 subsets/groups of cells or individual immune cells expressing various antibodies or TCRs. The methods of the present invention maximize immune cell recovery and provide a high degree of diversity.

[0197] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, intestinal lymphoid tissue, mucosal lymphoid tissue, spleen tissue, or any other. lymphoid tissue and tumors. T cells can be obtained from T cell lines and from autologous or allogeneic sources. T cells can be obtained from a single individual or a population of individuals, for example, a population of individuals where all suffer from the same disease, such as cancer or an infectious disease. In some embodiments, cells from the individual's circulating blood are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells harvested by apheresis or leukapheresis can be washed to remove a plasma fraction and load the cells into an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution is free of calcium and may be free of magnesium or many, if not all, divalent cations. As those skilled in the art will readily appreciate, the washing step can be carried out by methods known to those skilled in the art, such as using a "flow-through" semi-automatic centrifuge.

[0198] After washing, cells can be resuspended in various biocompatible buffers, such as, for example, Ca++/Mg++ free PBS. Alternatively, unwanted components of the apheresis sample can be removed and the cells directly resuspended in culture medium. In other embodiments, T cells are isolated from peripheral blood lymphocytes by erythrocyte lysis and PERCOLL™ gradient centrifugation. A specific subset of T cells, such as CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection methods. For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (eg DYNABEADS® M-450 CD3/CD28 T cell expander).

[0199] In some embodiments, enrichment of a T cell population by negative selection can be achieved with a combination of antibodies directed to surface markers unique to the negatively selected cells. One such method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry, which uses a set of monoclonal antibodies directed to cell surface markers present on cells that can be selected by negative selection. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Another way to prepare T cells for stimulation is a cell freezing step after a washing step, which does not require a monocyte removal step. The freeze-and-thaw step can provide a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population. After a washing step in which plasma and platelets are removed, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and would be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing media. This solution is diluted 1:1 with medium so that the final concentration of DMSO and HSA is 10% and 4%, respectively. The cells are then frozen to -80° C. at a rate of 1° C. per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0200] In some embodiments, a cell population is enriched from a sample. In some embodiments, cells are enriched for a particular cell group or cell subtype. In some embodiments, the cell population is enriched or contains T cells or B cells. In some embodiments, the cell population is enriched in or contains CD4+ or CD8+ cells. In some embodiments, the cell population is enriched in or contains central memory T cells, effector memory T cells, naive T cells, central memory stem T cells, effector T cells, and regulatory T cells. In some embodiments, the cell population is enriched in or contains memory B cells, naive B cells, or plasmablast B cells.

[0201] In some embodiments, immune cells can be selected based on the affinity of the cell's immune receptors for the selected target antigen or complex. In some aspects, affinity is related to the equilibrium constant for the reversible binding of two agents and is expressed as K _D . The affinity of the binding protein for the ligand, such as the affinity of an antibody for an epitope or such as the affinity for an MHC-peptide complex, can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar ( pM) or from about 100 nM to about 1 femtomolar (fM). The term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution.

[0202] In some embodiments, an epitope in some aspects refers to a portion of an antigen or other macromolecule capable of forming a binding interaction with the binding pocket of an antibody variable region or TCR. Such binding interactions may appear as intermolecular contact with one or more amino acid residues of one or more CDRs. Antigen binding may include, for example, a CDR3, a pair of CDR3s, or, in some cases, interactions of up to all six VH and VL chain CDRs. An epitope may be a linear peptide sequence (ie, "contiguous") or may be composed of non-contiguous amino acid sequences (ie, "conformational" or "discontinuous"). An antibody or TCR may recognize one or more amino acid sequences; therefore, an epitope may define more than one distinct amino acid sequence. In some aspects, the TCR may recognize one or more amino acid sequences or epitopes in the context of an MHC. Epitopes recognized by antibodies and TCRs can be determined by peptide mapping and sequencing sequencing methods well known to those skilled in the art. Binding interactions appear as intermolecular contacts with one or more CDR amino acid residues.

[0203] In some embodiments, reference to an immune receptor expressed on immune cells, such as an antibody or TCR having specific binding refers to a situation in which the antibody or TCR does not show any significant binding to a different type of molecule than the antigen containing an epitope recognized by an antibody or TCR. The term is also applicable when, for example, an antigen-binding domain is specific for a particular epitope that belongs to a set of antigens, in which case the selected antibody, TCR, or antigen-binding fragment thereof, carrying the antigen-binding domain, will be able to bind to different antigens bearing that epitope.

[0204] The terms "preferably binds" or "specifically binds" mean that antibodies, TCRs, or fragments thereof bind to an epitope with greater affinity than it binds unrelated amino acid sequences, and even if they cross-react with other polypeptides containing epitopes, they are not toxic at the concentrations for which they are formulated for human administration. In one aspect, such affinity is at least 1 fold greater, at least 2 fold greater, at least 3 fold greater, at least 4 fold greater, at least 5 fold greater, at least 6 times more at least 7 times more, at least 8 times more, at least 9 times more, 10 times more, at least 20 times more, at least 30 times more, at least 40 times more, at least 50 times more, at least 60 times more, at least 70 times more, at least 80 times more, at least 90 times more, at least 100 times more times greater or at least 1000 times greater than the affinity of the antibody, TCR or fragments thereof for non-specific amino acid sequences. The term "binding" refers to direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and/or hydrogen bonds under physiological conditions, and includes interactions such as salt and water bridges, as well as any other known means of binding.

[0205] In some embodiments, the term "binding" refers to a direct association between two molecules, e.g., due to covalent, electrostatic, hydrophobic, and ionic and/or hydrogen bonds under physiological conditions, and includes interactions such as salt and water bridges, as well as any other conventional means of binding.

[0206] In some embodiments, immune cells can be selected based on the affinity of an immuno receptor, such as a TCR cell, for a tetramer or other multimeric MHC peptide. In some embodiments, the term "tetramer" may refer to a complex containing four subunits associated with a single streptavidin molecule that can bind and thus identify a population of cells. The subunit may be an MHC-peptide complex. The subunit may be an MHC without an associated peptide. The subunit may be a B cell receptor antigen. The population of cells identified by the tetramer may be a population expressing a receptor, such as TCR or BCR, that binds to a subunit of the tetramer. The cell population may be antigen-specific T cells. The cell population may be antigen-specific B cells. The tetramer can be fluorescently labeled. As used herein, the term "MHC-peptide tetramer" may be used interchangeably with pMHC.

[0207] In some examples, immune cells can be selected based on affinity for an affinity oligonucleotide conjugate (see, for example, WO 2017/053905). Cells selected based on binding or recognition of a selected antigen or target complex, such as an affinity oligonucleotide conjugate, can be further isolated using the positive or negative selection methods described herein.

[0208] In some embodiments, immune cells from unimmunized human or non-human donors are used. An animal's naive repertoire (repertoire prior to antigen challenge) provides the animal with antibodies or TCRs that can bind with moderate affinity (K _A from about 1×10 ^-6 to 1×10 ^-7 M) to essentially any non-self molecule. Sequence diversity of antibody binding sites or TCRs is not encoded directly in the germline, but is provided in a combinatorial fashion by assembly from V gene segments. Immunization triggers any immune cell producing a combination of V _H- V _L or V _α- V _β or V _γ- V _δ that binds the immunogen, proliferates (clonal expansion) and secretes the appropriate antibody as noted above. However, the use of spleen cells and/or immune cells or other peripheral blood lymphocytes (PBL) from a non-immunized subject may provide a more accurate representation of the possible antibody or TCR repertoire and also allow the construction of a subsequent B-cell, T-cell antibody library or TCR library with using any kind of animal.

[0209] In some embodiments, the sample is saliva. In some embodiments, the sample is whole blood. In some embodiments, in order to obtain a sufficient number of polynucleotides for testing, the blood volume is at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45 or 50 ml. In some cases, to obtain sufficient nucleic acid for testing, the blood volume is at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10 , 20, 25, 30, 35, 40, 45 or 50 ml.

[0210] In some cases, the starting material is peripheral blood. Peripheral blood cells can be enriched for a particular cell type (eg, mononuclear cells; erythrocytes; CD4+ cells; CD8+ cells; immune cells; T cells, NK cells, or the like). Peripheral blood cells can also be selectively depleted for a particular cell type (eg, mononuclear cells; erythrocytes; CD4+ cells; CD8+ cells; immune cells; T cells, NK cells, or the like).

[0211] In some cases, the starting material may be a tissue sample containing hard tissue, with non-limiting examples including skin, brain, liver, lung, kidney, prostate, ovary, spleen, lymph node (including tonsil), thyroid, pancreas gland, heart, skeletal muscles, intestines, larynx, esophagus and stomach. In other cases, the starting material may be cells containing nucleic acids, immune cells and, in particular, B-cells or T-cells. In some cases, the starting material may be a sample containing nucleic acids from any organism from which genetic material can be obtained. In some cases, the sample is a liquid such as blood, saliva, lymph, or urine.

[0212] The sample may be taken from a subject with a disease. In some cases, the subject from which the carrier is taken may be a patient, for example, a cancer patient or suspected of having cancer. The subject may be a mammal, such as a human, male, or female. In some cases, the female may be pregnant. The sample may be a tumor biopsy. The biopsy may be performed, for example, by a healthcare professional, including a physician, physician assistant, nurse, veterinarian, dentist, chiropractor, medical assistant, dermatologist, oncologist, gastroenterologist, or surgeon.

[0213] In some cases, non-nucleic acid material may be removed from the starting material by enzymatic processing (such as protease digestion).

[0214] In some cases, blood can be collected in a device containing magnesium chelators, including, but not limited to, EDTA, and stored at 4°C. Optionally, a calcium chelator may be added, including but not limited to EGTA. In another case, a cell lysis inhibitor is added to the blood, including, but not limited to, formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, protein cross-linker, nucleic acid cross-linker, protein-nucleic acid cross-linkers, amino-reactive cross-linkers. -linkers, sulfhydryl-reactive cross-linkers, sulfhydryl-additive or disulfide-reducing cross-linkers, carbohydrate-reactive cross-linkers, carboxyl-reactive cross-linkers, photoreactive cross-linkers, or cleavable cross-linkers.

[0215] In some cases, where the extracted material contains single-stranded RNA, double-stranded RNA, or DNA-RNA hybrid, these molecules can be converted to double-stranded DNA using techniques known in the art. For example, reverse transcriptase can be used to synthesize DNA from RNA molecules. In some cases, converting RNA to DNA may require a preliminary ligation step to ligate the linker fragment to the RNA, allowing the use of universal primers to initiate reverse transcription. In other cases, for example, the poly-A tail of an mRNA molecule can be used to initiate reverse transcription. After converting RNA to DNA, the methods detailed herein can be used, in some cases, to further capture, select, label, or isolate the desired sequence.

[0216] Nucleic acid molecules include deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). Nucleic acid molecules can be synthetic or derived from natural sources. In one embodiment, nucleic acid molecules are isolated from a biological sample containing a variety of other components such as proteins, lipids, and non-matrix nucleic acids. The nucleic acid template molecules can be obtained from any cellular material isolated from an animal, plant, bacterium, fungus, or any other cellular organism. In certain embodiments, nucleic acid molecules are obtained from a single cell. Biological samples for use in the present invention include viral particles or preparations thereof. Nucleic acid molecules can be isolated directly from the body or from a biological sample obtained from the body, such as blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Any sample of tissue or body fluid y be used as a source of nucleic acid for use in the invention. Nucleic acid molecules can also be isolated from cultured cells, such as a primary cell culture or cell line. The cells or tissues from which the template nucleic acids are derived may be infected with a virus or other intracellular pathogen.

[0217] The sample can also be total RNA obtained or generated from biological material, cDNA library, viral or genomic DNA. In certain embodiments, nucleic acid molecules are associated/linked to other target molecules such as proteins, enzymes, substrates, antibodies, binding agents, beads, small molecules, peptides, or any other molecules. Typically, a nucleic acid can be extracted from a biological sample by various methods such as those described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, NY (2001). Nucleic acid molecules can be single stranded, double stranded, or double stranded with single stranded regions (eg stem and loop structures).

[0218] Methods for isolating DNA are well known in the art. The classical DNA isolation protocol is based on extraction using organic solvents such as a mixture of phenol and chloroform, followed by ethanol precipitation (J. Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, NY). Other methods include: salting out with DNA extraction (P. Sunnucks et al., Genetics, 1996, 144: 747-756; SM Aljanabi et al., Nucl. Acids Res. 1997, 25: 4692-4693), DNA extraction with trimethylammonium bromide salts (S. Gustincich et al., BioTechniques, 1991, 11: 298-302) and DNA extraction using guanidinium thiocyanate (JBW Hammond et al., Biochemistry, 1996, 240: 298-300). Various kits for DNA extraction from biological samples are commercially available (e.g., BD Biosciences Clontech (Palo Alto, CA): Epicentre Technologies (Madison, WI); Gentra Systems, Inc. (Minneapolis, MN); MicroProbe Corp. (Bothell, WA ); Organon Teknika (Durham, NC); and Qiagen Inc. (Valencia, CA)).

[0219] Methods for isolating RNA are also well known in the art (e.g., J. Sambrook et al., “Molecular Cloning: A Laboratory Manual” 1989, 211d Ed., Cold Spring Harbor Laboratory Press: New York), and kits are available for RNA extraction from biological fluids commercially (e.g., Ambion, Inc. (Austin, TX); Amersham Biosciences (Piscataway, NJ); BD Biosciences Clontech (Palo Alto, CA); BioRad Laboratories (Hercules, CA); Dynal Biotech Inc. ( Lake Success, NY); Epicentre Technologies (Madison, WI); Gentra Systems, Inc. (Minneapolis, MN); GIBCO BRL (Gaithersburg, MD); Invitrogen Life Technologies (Carlsbad, CA); MicroProbe Corp. (Bothell, WA) ; Organon Teknika (Durham, NC); Promega, Inc. (Madison, WI); and Qiagen Inc. (Valencia, CA)).

[0220] One or more samples may come from one or more sources. One or more samples may come from two or more sources. One or more samples may be from one or more subjects. One or more samples may come from two or more subjects. One or more samples may come from the same subject. One or more specimens may be from the same species. One or more specimens may be from different species. One or more subjects may be healthy. One or more subjects may have a disease, disorder, or condition.

[0221] In some embodiments, the sample is a fluid such as blood, saliva, lymph, urine, cerebrospinal fluid, semen, sputum, stool, or tissue homogenates.

[0222] The sample may be taken from a subject with an abnormal condition. In some embodiments, the subject from whom the carrier is taken may be a patient, such as a patient with cancer or suspected of having cancer. The subject may be a mammal, such as a human, and may be male or female. In some embodiments, the woman may be pregnant. The sample may be a tumor biopsy. The biopsy may be performed, for example, by a healthcare professional, including a physician, physician assistant, nurse, veterinarian, dentist, chiropractor, medical assistant, dermatologist, oncologist, gastroenterologist, or surgeon.

[0223] In some embodiments, the polynucleotides are linked to other target molecules, such as proteins, enzymes, substrates, antibodies, binding agents, beads, small molecules, peptides, or any other molecules. In some embodiments, the polynucleotides are not associated with a solid support. Nucleic acids can be extracted from a biological sample in various ways (Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, NY (2001)).

[0224] In some embodiments, cell suspensions may be prewarmed prior to analysis. In some embodiments, cell suspensions are heated just prior to emulsion generation (described in section B.2 below) to a temperature and for a sufficient duration to increase DNA polymerase activity within the cell and to minimize undesirable effects such as RNA degradation. Thus, cells are heated to optimize RNA yield according to the methods presented here. In some examples, the cells are heated to about 30-70°C, e.g., 30 to 60°C, 25 to 60°C, 30 to 60°C, 40 to 60°C, 45 to 55°C, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minutes. After heating the cells, the cell suspension can be kept at room temperature or placed on ice for 30 seconds to 4 hours, e.g. 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours 3, 5 hours or 4 hours to emulsify.

[0225] The number of samples may contain at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more samples. The number of samples may contain at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more samples. The number of samples may contain at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 samples, 9000 or 10000 samples, or 100000 samples, or 1000000 or more samples. The number of samples may contain at least about 10,000 samples.

[0226] One or more polynucleotides of the first sample may differ from one or more polynucleotides of the second sample. One or more polynucleotides of the first sample may differ from one or more polynucleotides of multiple samples. One or more polynucleotides in a sample may contain sequences with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In some embodiments, one or more sample polynucleotides may differ by less than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide or base pair. A plurality of polynucleotides in one or more samples from a plurality of samples may contain two or more identical sequences. At least about 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%, 98%, 99% or 100% of the total polynucleotides in one or more of the multiple samples may contain the same sequence. A plurality of polynucleotides in one or more samples from a plurality of samples may contain at least two different sequences. At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of all polynucleotides in one or more samples from multiple samples may contain at least two different sequences. In some embodiments, one or more polynucleotides are variants of each other. For example, one or more polynucleotides may contain single nucleotide polymorphisms or other types of mutations. In another example, one or more polynucleotides are splicing variants.

[0227] The first sample may contain one or more cells, and the second sample may contain one or more cells. One or more cells of the first sample may be of the same type as one or more cells of the second sample. One or more of the cells in the first sample may be of a different cell type than one or more different cells belonging to the plurality of samples.

[0228] Many samples can be obtained in parallel. Multiple samples can be taken at the same time. A plurality of samples may be obtained sequentially. A plurality of samples may be obtained over several years, such as 100 years, 10 years, 5 years, 4 years, 3 years, 2 years, or 1 year after receiving one or more different samples. One or more samples can be obtained within about one year after receiving one or more different samples. One or more samples may be received within 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months or 1 month from receipt of one or more various samples. One or more samples can be received within 30 days, 28 days, 26 days, 24 days, 21 days, 20 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days , 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or one day after receiving one or more different samples. One or more samples may be obtained within approximately 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour from their receipt of one or more different samples. One or more samples may be taken within about 60 seconds, 45 seconds, 30 seconds, 20 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second of one or more different samples being taken. One or more samples can be obtained within less than one second after receiving one or more different samples.

[0229] Different sample polynucleotides may be present in the sample at different concentrations or amounts (eg, different numbers of molecules). For example, the concentration or amount of one polynucleotide may be greater than the concentration or amount of another polynucleotide in the sample. In some embodiments, the concentration or amount of at least one polynucleotide in a sample may be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more times more than the concentration or amount of at least one other polynucleotide in the sample. In another example, the concentration or amount of one polynucleotide is less than the concentration or amount of another polynucleotide in the sample. The concentration or amount of at least one polynucleotide in a sample may be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 , 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more times less than the concentration or amount in at least one other polynucleotide in the sample.

[0230] In some embodiments, two or more samples may contain different amounts or concentrations of polynucleotides. In some embodiments, the concentration or amount of one polynucleotide in one sample may be greater than the concentration or amount of the same polynucleotide in another sample. For example, a blood sample may contain a higher amount of a particular polynucleotide than a urine sample. Alternatively, one sample may be divided into two or more samples. Such samples may contain different amounts or concentrations of the same polynucleotide. The concentration or amount of at least one polynucleotide in one sample may be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25. 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more times greater than the concentration or the amount of the same polynucleotide in another sample. Alternatively, the concentration or amount of one polynucleotide in one sample may be less than the concentration or amount of the same polynucleotide in another sample. For example, the concentration or amount of at least one polynucleotide in one sample may be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more times less than concentration or the amount of the same polynucleotide in another sample.

2. Obtaining droplets and barcoding individual cells

[0231] For single cell barcoding using a carrier barcode and a molecular barcode, carriers such as water-in-oil emulsions can be designed such that the resulting carriers contain 1 cell or less per carrier . Media can be created such that the resulting media also contains 1 media barcode per media. Carriers can be designed such that the resulting carriers also contain 1 molecular barcode polynucleotide per carrier. The carriers can be designed such that the resulting carriers also contain two or more molecular barcoded polynucleotides per carrier. Cells/carriers can be subjected to a single code RNA or DNA barcoding protocol as described herein, and the carrier barcode and one or more molecular barcodes of each carrier can be fused to a target of interest, such as a cellular polynucleotide. In some embodiments, polynucleotides with a matching carrier barcode can be fused to cellular components present in the same carrier, such as one or more molecular barcoded polynucleotides. After sequencing, the carrier barcode and molecular barcode can be used to mathematically determine which RNA (or DNA) came from which cell. In some embodiments, carriers, such as water-in-oil emulsions, can be formulated such that the resulting emulsions contain 1 cell or more per emulsion. In some embodiments, water-in-oil emulsions can be formulated such that the resulting emulsions contain 1 carrier barcode polynucleotide and two or more molecular barcode polynucleotides per carrier. In some embodiments, the carriers can be designed such that the resulting carriers contain more than one carrier barcode polynucleotide and two or more molecular barcode polynucleotides per carrier. In some embodiments, the carrier barcode and the molecular barcode may be incorporated into carriers in solution. In some embodiments, the carrier barcode and the molecular barcode may be entered into carriers as long as they are not attached to a solid support such as beads. Typical carriers are well, emulsion, drop and microcapsule.

[0232] In some aspects, individual cells can be enclosed within an emulsion that can act as a compartment (eg, carrier). Cells can be lysed and cell transcripts can be barcoded. Each of the transcripts can be linked to a molecular or carrier barcode such that if two or more RNA transcripts are found with the same carrier barcode, they can be diagnosed as originating from the same parent cell. . This can be applied to many different types of sequences. One particular application would be linking VH and VL or Vα and Vβ or Vγ and Vδ antibody chains to TCR sequences.

[0233] One or more individual cells can be enclosed in one or more emulsions in the presence of a carrier barcode and molecular barcodes, so that one carrier, such as a drop, from one or more emulsions can contain a maximum of 1 cell or less . Cells can be chemically lysed with the buffer contained in the emulsion or by thawing, thereby releasing the contents of the cell into the emulsion.

[0234] Single cell RNA can be transcribed into cDNA by reverse transcription. The reverse transcription reaction can be carried out with a reverse transcriptase that has non-template terminal transferase activity, adds about 3 cytosine residues, as described above. All reverse transcription buffers, enzymes and nucleotides may be present when the emulsion is formed. In some embodiments, the primer is a generalized sequence (such as a polynucleotide containing the poly dT sequence) to target the entire mRNA. In some embodiments, DNA may be used. In some embodiments, more than 2 RNAs can serve as targets.

[0235] In some embodiments, the carrier barcode may be associated with RNA during reverse transcription. In some embodiments, the implementation of the molecular barcode can be associated with RNA during reverse transcription. In some embodiments, the carrier barcode and the molecular barcode can be linked to RNA during reverse transcription. Dividing a sample of multiple cells into small reaction volumes, combined with barcoding of polynucleotides with a molecular barcode and a carrier barcode belonging to or originating from a single cell from a plurality of cells, can provide high throughput. sequencing a repertoire of sequences such as biomarker sequences.

[0236] Dividing a sample of multiple cells into small reaction volumes or carriers containing one or more cells, in combination with barcoding of polynucleotides with a molecular barcode and a carrier barcode belonging to or originating from a single cell from a plurality of cells, can provide high throughput ability. sequencing a repertoire of sequences, such as sequences representing the percentage of an organism's transcriptome. For example, the sequence repertoire may contain a plurality of sequences representing at least about 0.00001%, 0.00005%, 0.00010%, 0.00050%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9 %, 10%, 15%, 20%, 30%, 35%, 40% 45, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 98%, 99%, or 100% of an organism's transcriptome.

[0237] Separation of a sample of immune cells into small reaction volumes or carriers containing one or more immune cells, in combination with barcoding of polynucleotides with a molecular barcode and a carrier barcode belonging to or derived from a single immune cell from a plurality of immune cells can ensure high throughput. sequencing of the repertoire of heavy and light chain sequences. These methods can also allow heavy and light chain assignment after sequencing based on barcoded sequences. Dividing the sample into small reaction volumes, as described here, can also allow the use of reduced amounts of reagents, thereby reducing the material costs of the assay.

[0238] In some cases, the reverse transcription reaction and/or the amplification reaction (eg, PCR) is carried out in drops, such as digital drop PCR. In certain aspects, the invention provides fluid compartments or carriers to contain all or part of the target material. In some embodiments, the compartment or carrier is a droplet. Although "drops" are referred to throughout the specification, these terms are used interchangeably with fluid containment compartment and fluid delineation unless otherwise noted. The carrier may comprise or consist of such a fluid compartment with defined fluid separators/boundaries. Unless otherwise indicated, the term "droplet" is used for convenience, but any fluid-restricting compartment, baffle, or fluid compartment may be used. Drops used herein may include emulsion compositions (or mixtures of two or more immiscible liquids) such as those described in US Pat. No. 7,622,280. Drops may be generated by the devices described in WO/2010/036352. As used herein, the term "emulsion" may refer to a mixture of immiscible liquids (such as oil and water). Oil and/or water-oil emulsions make it possible to separate reaction mixtures within aqueous droplets. Emulsions may contain water droplets in a continuous oil phase. The emulsions provided here may be oil-in-water emulsions where the droplets are oil droplets in a continuous aqueous phase. The drops described herein are intended to prevent mixing between compartments, where each compartment prevents its contents from evaporating and/or merging with the contents of other compartments.

[0239] The mixtures or emulsions described herein may be stable or unstable. Emulsions may be relatively stable and have minimal coalescence. Coalescence occurs when small droplets combine to form progressively larger droplets. In some cases less than 0.00001%, 0.00005%, 0.00010%, 0.00050%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9% or 10% droplets obtained with a drop generator merge with other drops. Emulsions may also have limited flocculation, a process by which the discontinuous phase emerges from suspension as loose flocculent aggregates.

[0240] Droplets can be obtained having an average diameter of about, slightly less than, or slightly greater than at least about 0.001, 0.01, 0.05, 0.1, 1, 5, 10, 20, 30, 40 , 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, 400 or 500 µm. The droplets may have an average diameter of from about 0.001 to about 500, from about 0.01 to about 500, from about 0.1 to about 500, from about 0.1 to about 100, from about 0.01 to about 100, or from about 1 to about 100 µm. Microfluidic methods for producing emulsion droplets using microchannel cross-flow focusing or physical agitation are known to produce either monodisperse or polydisperse emulsions. The drops may be monodisperse drops or carriers. The droplets can be formed such that the droplet size does not change by more than plus or minus 5% of the average droplet size. In some cases, the droplets are generated such that the droplet size does not change by more than plus or minus 2% of the average droplet size. The Droplet Generator can generate a population of droplets from a single sample, with none of the droplets differing in size by more than plus or minus approximately 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5% , 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9 %, 9.5% or 10% of the average size of the entire population of drops.

[0241] A drop or carrier can be formed by passing the oil phase through an aqueous sample. The aqueous phase may contain a buffer solution and amplification reaction reagents, including cells, nucleotides, nucleotide analogs, molecular barcoded polynucleotides, carrier barcoded polynucleotide primers, template nucleic acids, and enzymes such as DNA polymerase, RNA polymerase and/or reverse transcriptase. In some embodiments, the aqueous phase may contain a cell lysis reagent, such as a cell lysis chemical.

[0242] The aqueous phase may contain a buffered solution and reagents for carrying out the amplification reaction on a solid substrate, such as a bead, or without it. The buffered solution may contain about, more than about, or less than about 1, 5, 10, 15, 20, 30, 50, 100, or 200 mM Tris. In some cases, the potassium chloride concentration may be about, more than about, or less than about 10, 20, 30, 40, 50, 60, 80, 100, 200 mM. The buffered solution may contain approximately 15 mm Tris and 50 mm KCl. Nucleotides may contain deoxyribonucleotide triphosphate molecules, including dATP, dCTP, dGTP, and dTTP, at concentrations of about, more than about, or less than about 50, 100, 200, 300, 400, 500, 600, or 700 μM each. In some cases, dUTP is added in the aqueous phase to a concentration of about, more than about, or less than about 50, 100, 200, 300, 400, 500, 600, or 700, 800, 900, or 1000 µM. In some instances, magnesium chloride or magnesium acetate (MgCl is added to the aqueous phase at a concentration of about, more than about, or less than about 1.0, 2.0, 3.0, 4.0, or 5.0 mM). The MgCl concentration may be about 3.2 mM. In some cases magnesium or magnesium acetate is used. In some cases, magnesium sulfate is used.

[0243] A non-specific blocking agent can be used, such as BSA or bovine gelatin, where the gelatin or BSA is present in a concentration range of about 0.1-0.9% w/v. Other possible blocking agents may include beta-lactoglobulin, casein, milk powder, or other common blocking agents. In some cases, the preferred concentrations of BSA and gelatin are about 0.1% w/v.

[0244] The aqueous phase amplification primers may have a concentration of about, more than about, or less than about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0, 6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5. 1.7 or 2.0 µM. The primer concentration in the aqueous phase can be from about 0.05 to about 2, from about 0.1 to about 1.0, from about 0.2 to about 1.0, from about 0.3 to about 1.0, from about 0.4 to about 1.0, or about 0.5 to about 1.0 μM. The primer concentration may be approximately 0.5 μM. Suitable ranges for target nucleic acid concentrations in PCR include, but are not limited to, from about 1 μg to about 500 ng.

[0245] In some cases, the aqueous phase may also contain additives, including, but not limited to, non-specific background / blocking nucleic acids (for example, salmon sperm DNA), biopreservatives (for example, sodium azide), PCR enhancers (for example, betaine, trehalose and others) and inhibitors (for example, RNase inhibitors). Other additives may include, for example, dimethyl sulfoxide (DMSO), glycerol, betaine (mono) hydrate (N,N,N-trimethylglycine=[carboxymethyl]trimethylammonium), trehalose, 7-deaza-2'-deoxyguanosine triphosphate (dC7GTP or 7- deaza-2′-dGTP), BSA (bovine serum albumin), formamide (methanamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (e.g. tetraethylammonium chloride (TEA-C1) and tetrapropylammonium chloride (TPrACl), nonionic detergents (e.g. , Triton X-100, TWEEN® 20, Nonidet P-40 (NP-40)) or PREXCEL-Q In some cases, the aqueous phase may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different additives In other cases, the aqueous phase may contain at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different additives.

[0246] In some cases, the non-ionic ethylene oxide/propylene oxide block copolymer may be added to the aqueous phase at a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6 %, 0.7%. 0.8%, 0.9% or 1.0%. Common biosurfactants include nonionic surfactants such as Pluronic F-68, Tetronics and Zonyl FSN. Pluronic F-68 may be present at a concentration of about 0.5% w/v.

[0247] In some cases, magnesium sulfate can be replaced with magnesium chloride at similar concentrations. A wide range of common commercial PCR buffers from various vendors can be replaced with a buffer solution.

[0248] This refers to carriers that have a liquid-like or solid-like interface with the surrounding phase. For example, in some embodiments, the implementation of the emulsion can be prepared to obtain highly monodisperse droplets serving as carriers, and having a liquid-like interfacial film, which can be converted by heating into microcapsules having a solid-like interfacial film; such microcapsules can act as bioreactors capable of retaining their contents during a reaction such as PCR amplification. The transformation of the form of the carrier into the form of a microcapsule can occur when heated. For example, such conversion may occur at a temperature greater than about 50°C, 60°C, 70°C, 80°C, 90°C or 95°C. In some cases, this heating is carried out using a thermal cycler. Liquid or mineral oil can be added during the heating process to prevent evaporation. Excess continuous phase oil may or may not be removed prior to heating. Biocompatible capsules may be resistant to coalescence and/or flocculation over a wide range of thermal and mechanical treatments. Once converted into a capsule, they may be stored at about, more than about, or less than about 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C. Such capsule-shaped carriers may be useful for biomedical applications such as computer-processed stable encapsulation of macromolecules, in particular aqueous biological fluids containing a mixture of nucleic acids or protein, or both; delivery of medicines and vaccines; biomolecular libraries; applications for clinical imaging and others.

[0249] Microcapsules may contain one or more polynucleotides and may resist coalescence, especially at high temperatures. Accordingly, PCR amplification reactions can occur at very high densities (eg, number of reactions per unit volume). In some cases, more than 100,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 5,000,000, or 1,000,000 individual reactions may occur per ml. In some cases, reactions occur in a single well, such as a well of a microtiter plate, without mixing between reaction volumes. The microcapsules may also contain other components necessary to enable reverse transcription, primer extension and/or PCR reaction, such as primers, probes, dNTPs, DNA or RNA polymerases, etc. Such capsule-shaped carriers exhibit resistance to coalescence and flocculation over a wide range of thermal and mechanical treatments.

[0250] In some cases, the amplification step is performed by performing a digital PCR, such as microfluidic digital PCR or spot digital PCR.

[0251] In some embodiments, the carriers may be drops. Droplets can be produced using microfluidic systems or devices. As used herein, the prefix "micro" (eg, "microchannel" or "microfluidic") generally refers to features or articles having a width or diameter of less than about 1 mm and less than about 100 µm (micrometers). In some cases, the element or product contains a channel through which a liquid can flow. In addition, "microfluidic" as used herein refers to a device, apparatus, or system that includes at least one micro-scale channel.

[0252] Microfluidic systems and devices have been described in a variety of contexts, typically in the context of miniature laboratory (eg, clinical) analysis. Other uses have also been described. For example, in International Patent Publication Nos. WO 01/89788; WO2006/040551; WO2006/040554; WO2004/002627; WO2008/063227; WO2004/091763; WO 2005/021 151; WO2006/096571; WO2007/089541; WO 2007/081385 and WO 2008/063227.

[0253] The droplet typically includes an amount of the first carrier fluid in the second carrier fluid. Any technology known in the art for forming droplets can be used in accordance with the methods of the invention. An exemplary method includes flowing a carrier fluid stream containing a target material (eg, an immune cell) such that it intersects two oppositely directed carrier fluid streams. The carrier fluid is not miscible with the carrier fluid. The intersection of the sample liquid with two oppositely directed streams of the current carrier liquid results in the separation of the sample liquid into separate droplets of the sample, which can serve as carriers containing the target material.

[0254] The carrier fluid can be any fluid that is immiscible with the carrier fluid. An example of a carrier fluid is oil. In certain embodiments, the implementation of the carrier fluid includes a surfactant.

[0255] The same method can be used to create single droplets or carriers that contain other reagents, such as reagents for an amplification reaction such as polymerase chain reaction (PCR) or non-PCR based amplification reactions such as multistrand displacement amplification or other methods known to the person skilled in the art. Suitable reagents for carrying out PCR-based amplification reactions are known to those skilled in the art and include, but are not limited to, DNA polymerases, forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and one or more buffers.

[0256] In some embodiments, fluid compartments are formed by providing a first fluid septum (e.g., a droplet) containing a target material (e.g., an immune cell and/or a solid support such as a bead) and a second fluid (e.g., a fluid stream or within the drops ). The first and second liquids are combined to form a drop that can serve as a carrier for the methods described. Merging can be achieved by applying an electric field to two fluids. In some embodiments, the second liquid contains reagents for carrying out an amplification reaction, such as a polymerase chain reaction or an amplification reaction.

[0257] Increased mechanical stability may be useful for microfluidic manipulations and high stress fluid processing (eg, in microfluidic capillaries or 90 degree fluid path rotations, eg, in valves). Before and after heat-treated drops or capsules can be mechanically resistant to standard pipetting and centrifugation.

3. Reverse transcription

[0258] In some cases, target polynucleotides are obtained from RNA, such as mRNA, by reverse transcription. In some instances, target polynucleotides are derived from DNA by primer extension, such as using a polymerase. During the reverse transcription reaction, cellular RNA, such as mRNA, is reverse transcribed to produce complementary DNA (cDNA), and a unique molecular barcode is added to each cDNA to generate a barcoded single stranded polynucleotide complementary to the cellular transcript. Such barcoded single strand polynucleotides can be generated for each transcript in the transcriptome.

[0259] The methods described herein can be used in conjunction with PCR with a reverse transcription reaction (reverse PCR with transcription). For example, reverse transcription and PCR can be carried out in two different steps. First, a cDNA copy of an mRNA sample can be synthesized using either the dT polynucleotide primer, a sequence-specific primer, a universal primer, a mixture of random hexamer oligonucleotide primers, or any primer described herein. In some embodiments, a cDNA copy of an RNA can be created using a mixture of primers, such as a sequence-specific primer, and a mixture of random hexameric oligonucleotide primers, for example, to capture specific target RNA molecules of a cell, in addition to a collection of polynucleotides that substantially match transcriptome of the same cell.

[0260] Reverse transcription and PCR can be performed in the same closed reaction vehicle. For example, multiple primers, one or more reverse transcription primers, and two or more PCR primers can be used in the same closed carrier. The reverse transcription primer(s) can bind to the mRNA at the 3' position of the first PCR amplicon. In some embodiments, the PCR conditions can be modified to substantially restrict amplification by the first adapter or pool of first adapters using their specific primers and to restrict amplification of the larger molecular barcode cDNA. Although not essential, the reverse transcription primer(s) may include RNA residues or modified analogs thereof, such as 2'-O-methylated RNA bases, which will not form a substrate for RNase H when hybridized to mRNA.

[0261] The temperature for carrying out the reverse transcription reaction depends on the reverse transcriptase used. In some instances, a thermostable reverse transcriptase is used and the reverse transcription reaction is carried out at about 37 to about 75°C, at about 37 to about 50°C, at about 37 to about 55°C, at about 37 to about 60°C, at about 55 to about 75°C, at about 55 to about 60°C, at about 37°C, or at about 60°C. In some cases, reverse transcriptase is used, which transfers 3 or more non-template terminal nucleotides to the end of the transcribed product.

[0262] The reverse transcription reaction and the PCR reaction described herein can be carried out in various formats known in the art, such as tubes, microtiter plates, microfluidic medium, or preferably drops.

[0263] The reverse transcription reaction can be carried out in volumes ranging from 5 μl to 100 μl, or in reaction volumes from 10 μl to 20 μl. In drops, reaction volumes can range from 1 pl to 100 nl, or from 10 pl to 1 nl. In some cases, the reverse transcription reaction is carried out in a drop having a volume of about 1 nl or less.

[0264] Target polynucleotides, such as RNA, can be reverse transcribed into cDNA using one or more reverse transcription primers. One or more reverse transcription primers may contain a region complementary to a region of the RNA, such as a constant region (eg, a heavy or light chain constant region or a polyA mRNA tail). In some embodiments, the reverse transcription primers may comprise a first reverse transcription primer with a region complementary to the constant region of the first RNA and a second reverse transcription primer with a region complementary to the constant region of the second RNA. In some embodiments, the reverse transcription primers may comprise a first reverse transcription primer with a region complementary to the constant region of the first RNA, and one or more reverse transcription primers with a region complementary to the constant region of one or more RNAs, respectively.

[0265] In some embodiments, reverse transcription primers can be modified to minimize artifact generation by exponentially amplifying primer-dimer or primer-template switch products in the reaction. In some embodiments, reverse transcription primers are modified by adding one or more 2'-O-methylated primer bases. In some embodiments, one or more 2'-O-methylated bases are located near the center of the primer sequence. Such modified primers are typically used in reactions involving DNA polymerase that cannot include bases opposite the 2'O-methyl modified residue. Representative 2'O-methyl modified primers are shown in SEQ ID NOS: 12-22).

[0266] In some embodiments, the reverse transcription primers are a mixture of random hexameric oligonucleotides. Such primers can bind RNA at random locations, thereby initiating a reverse transcription reaction of unknown sequences. In such examples, a sufficient number of random hexamer primers are used to effect reverse transcription of essentially the cell's transcriptome. Thus, in such embodiments, a collection of polynucleotides is created, such as a collection of cDNA polynucleotides, corresponding to the transcriptome of a cell.

[0267] In some embodiments, the reverse transcription primers do not contain a barcode.

[0268] The reverse transcription primers may further comprise a region that is not complementary to the RNA region. In some embodiments, the non-complementary region of the RNA is at the 5' position relative to the region of the primers that is complementary to the RNA. In some embodiments, the region that is not complementary to the RNA region is located at the 3' position relative to the primer region that is complementary to the RNA. In some embodiments, the non-complementary region of the RNA is the 5' overhang. In some embodiments, the region that is not complementary to the RNA region contains a priming site for amplification and/or sequencing reaction, such as an adapter. Using one or more of the primers described herein, the RNA molecules are reverse transcribed using suitable reagents known in the art.

[0269] In specific embodiments, the reverse transcriptase may contain non-template terminal transferase activity. When a reverse transcriptase comprising a non-template terminal transferase activity reaches the end of the template, it may add three or more non-template residues, such as three or more non-template cytosine residues. In some embodiments, Superscript II™ reverse transcriptase is used for this purpose. In some embodiments, Maxima™ reverse transcriptase is used for this purpose. In some embodiments, Protoscript II™ reverse transcriptase is used for this purpose. In some embodiments, Maloney murine leukemia virus (MMLV-RT) reverse transcriptase is used for this purpose. In some embodiments, HighScriber™ reverse transcriptase is used for this purpose. In some embodiments, a terminal deoxynucleotidyl transferase is used for this purpose. In some embodiments, avian myeloblastosis virus (AMV) reverse transcriptase is used for this purpose. Any reverse transcriptase capable of transcribing RNA that has non-template terminal transferase activity can be used. Any reverse polymerase capable of transcribing RNA that has non-template terminal transferase activity can be used. Any reverse polymerase capable of transcribing DNA that has non-template terminal transferase activity can be used.

[0270] Reverse transcription reactions such as those described above can be carried out in the presence of a 3' labeled polynucleotide. The 3'-labeled polynucleotide can be a polynucleotide used to add nucleic acids to the 3' end of a target polynucleotide, such as a cDNA. A 3'-labeled polynucleotide may be a polynucleotide used as a template for adding nucleic acids to the 3' end of a target polynucleotide, such as cDNA. A 3' labeled polynucleotide may be a polynucleotide that hybridizes to the 3' end of a target polynucleotide, such as cDNA. A 3'-labeled polynucleotide may be a polynucleotide that contains a 3' region, such as a 3' end region, that hybridizes to the 3' end of a target polynucleotide, such as cDNA. For example, a 3'-labeled polynucleotide may contain a segment, such as a segment that anneals to three or more non-template residues. In some embodiments, the 3' labeled polynucleotide is a molecular barcode polynucleotide. In some embodiments, the implementation of the 3'-labeled polynucleotide may contain a molecular barcode. In some embodiments, the 3' labeled polynucleotide may contain 3' riboguanosine residues or analogs thereof at the 3' end (rGrGrG) (RNA bases) that are complementary and anneal to a strand produced by a reverse transcription enzyme. In some embodiments, three or more guanine residues can be used instead of riboguanosine (a DNA nucleotide instead of an RNA nucleotide). In some embodiments, the 3'-labeled polynucleotide may contain 1 or 2 riboguanosine residues at the 3' end and a riboguanosine residue or analog thereof at the 3' end (rGrGG) that are complementary and anneal to the strand resulting from the work. reverse transcription enzyme.

[0271] When annealing a 3'-labeled polynucleotide with a CCC cDNA strand, reverse transcriptase can continue to extend the cDNA to the labeled polynucleotide, thereby attaching the molecular barcode or its complement to the target population of polynucleotides, such as cDNA, in a reaction. For example, a 3' labeled polynucleotide can be a polynucleotide that contains a 5' to 3' region that hybridizes to the 3' end of the target polynucleotide. The 5' to 3' region that hybridizes to the 3' end of the target polynucleotide may contain a region that is not complementary to the target polynucleotide, such as cDNA. The 5' to 3' region that hybridizes to the 3' end of the target polynucleotide may contain a molecular barcode. The 5' to 3' region that hybridizes to the 3' end of the target polynucleotide may contain a region complementary to the carrier barcode polynucleotide or its complement. In other experiments, matrix switching can be done in separate reactions. For example, a 3' labeled polynucleotide may be added after the reverse transcription reaction and enzymes such as reverse transcriptase or polymerase may be used to extend to the labeled polynucleotide. Since a labeled polynucleotide may contain a unique degenerate molecular barcode on each molecule in the carrier, each cDNA in the carrier may be uniquely labeled with a molecular barcode. In some embodiments, the template switch may be performed simultaneously with the reverse transcription reaction.

[0272] The reverse transcription reaction can be carried out in the presence of a 3' labeled polynucleotide. The 3'-labeled polynucleotide may contain a P7 segment which may be used to anneal the sequencing primer. The 3'-labeled polynucleotide may contain a carrier barcode or a molecular barcode. The 3' labeled polynucleotide may contain 3' riboguanosine residues at the 3' end (rGrGrG) (RNA bases) which may be complementary and anneal to the strand produced by the reverse transcription enzyme. Thus, the carrier barcode and the molecular barcode can be attached to the terminal end of the cDNA in the same emulsion by reverse transcription enzymes. In some embodiments, guanine residues can be used instead of riboguanosine (a DNA nucleotide instead of an RNA nucleotide). After annealing the 3'-labeled polynucleotide to the CCC cDNA strand, reverse transcriptase continues to extend the cDNA to the 3'-labeled polynucleotide, thereby creating a barcoded molecular label for all cDNA in the reaction. After annealing the 3'-labeled polynucleotide into the region of the molecular barcoded cDNA, reverse transcriptase or polymerase continues to extend the molecular-barcoded cDNA to another 3'-labeled polynucleotide, thereby creating a carrier barcode for all cDNA in the reaction.

[0273] In some embodiments, the template switch may be performed in a separate reaction rather than at the same time that the reverse transcription reaction may be performed. In some embodiments, a 3' labeled polynucleotide can be added after the reverse transcription reaction and enzymes such as reverse transcriptase or polymerase can be used to extend to the labeled polynucleotide in a similar manner. Because a 3'-labeled polynucleotide can carry a unique degenerate molecular barcode on each individual molecule, each cDNA can be uniquely labeled with a molecular barcode. Because a 3'-labeled polynucleotide can contain the same degenerate carrier barcode on every single molecule from a single carrier, each cDNA can be labeled with a carrier barcode unique to the carrier.

[0274] In some embodiments, a template switch molecule, such as a template switch oligonucleotide containing a barcode (eg, a molecular barcode), may include modified bases to minimize artifact generation. In some embodiments, a template-switched oligonucleotide may contain 2'-deoxyuridine, which can be reverse transcribed but cannot be copied by DNA polymerase. In some embodiments, riboguanosine may be included in a template switch oligonucleotide. In some embodiments, a template-switching oligonucleotide can be modified at the 3' end to prevent extension by reverse transcriptase or DNA polymerase. Such modifications include 3'-deoxy, 3'-phosphate, 3'-amino and 3'-alkyl modification to block primer extension.

4. Polymerase chain reaction (PCR)

[0275] After performing reverse transcription reactions of RNA molecules, the resulting single barcode cDNA molecules can be simultaneously barcoded with a carrier barcode and amplified by one or more PCR reactions to produce one or more amplicons. In some examples, PCR is used to generate a double-barcoded cDNA strand that contains a coding sequence corresponding to the mRNA transcript sequence and is complementary to the cDNA strand generated during the reverse transcriptase reaction. Enzymes and primer designs for PCR are known, and non-limiting examples of such reagents are described here. Any of these reagents may be selected and used for PCR in the methods provided.

[0276] In some cases, the PCR reaction occurs in a drop having a reaction volume in the range of 1pl to 100nl, preferably 10pl to 1nl. In some cases, the PCR reaction is carried out in a drop, the volume of which is about 1 nl or less. In some cases, the reverse transcription reaction and the PCR reaction are carried out in the same drop with a volume of the reaction mixture from 1pl to 100 nl or from 10pl to 1 nl. In some cases, the reverse transcription reaction and the PCR reaction are carried out in a drop having a volume of about 1 nl or less, or a volume of about 1 μl or less. In some cases, the reverse transcription reaction and the PCR reaction are carried out in different drops. In some cases, the reverse transcription reaction and the PCR reaction are carried out in a plurality of drops, each of which has a reaction volume in the range from 1 pl to 100 nl or from 10 pl to 1 nl. In some cases, the reverse transcription reaction and the PCR reaction are carried out in multiple drops, each of which has a volume of about 1 nl or less.

[0277] In some cases, the first PCR reaction occurs in the first drop, the reaction volume of which is in the range from 1 pl to 100 nl, preferably from 10 pl to 1 nl, and the second PCR reaction takes place in the second drop, the reaction volume of which is 1 pl up to 100 nl, preferably from 10 pl to 1 nl. In some cases, the first PCR reaction occurs in the first drop, which is about 1 nl or less in volume, and the second PCR reaction is in the second drop, which is about 1 nl or less in volume.

[0278] In some cases, the first PCR reaction and the second PCR reaction are carried out in a plurality of drops, each having a reaction volume in the range of 1pl to 100 nL or 10pl to 1 nL. In some cases, the first PCR reaction and the second PCR reaction are carried out in a plurality of drops, each of which has a volume that is about 1 nl or less.

[0279] In some embodiments of the methods provided herein, the reaction conditions can be modified to enhance the amplification of selected sequences and minimize the amplification of other sequences. Such modifications may include temperature changes, such as melting points, during PCR thermal cycling. For example, "cold" PCR cycles can be used to selectively amplify shorter oligonucleotides. "Cold" PCR cycles differ from "hot" PCR cycles in their denaturation temperature. "Cold" PCR cycles affect denaturation at a lower temperature to preferentially amplify shorter sequences that denature more easily than longer double-stranded sequences. Thus, "cold" PCR cycles are used to amplify shorter sequences while limiting the amplification of longer sequences. In some embodiments, a combination of "cold" cycles and "hot cycles" is used to create the desired amplicons.

[0280] In some embodiments, the duration of the steps of denaturation, priming and/or extension of PCR can be modified to selectively or preferentially amplify certain sequences in the reaction volume. In some examples, primers used for PCR amplification may be selected or modified to reduce or enhance PCR amplification under certain conditions. In some embodiments, thermolabile auxiliary groups can be added to the 3' end of most bases via phosphotriester linkages to render the oligonucleotide primers inactive at lower temperatures but active after exposure to higher temperatures. In some embodiments, oligonucleotides linked to a thermolabile auxiliary group at the 3' end are used in the provided methods, whereby the oligonucleotides are not capable of primer extension at lower temperatures, such as temperatures at which reverse transcriptase reactions occur, but become active when exposed to a higher temperature, such as before or during PCR.

[0281] After performing reverse transcription reactions of RNA molecules or primer extension of genomic molecules, an oligonucleotide containing a carrier barcode is amplified by polymerase chain reaction to obtain multiple copies that are attached to polynucleotides with a molecular barcode. In some examples, oligonucleotides containing a carrier barcode are amplified using primers modified by adding a heat-labile auxiliary group at the 3' end to prevent primer extension during the reverse transcriptase reaction, but allow primer extension and subsequent amplification during PCR. In some embodiments, the implementation of the amplification of the oligonucleotide containing the barcode of the carrier, was carried out using "cold" thermal cycling, as described here.

[0282] After performing reverse transcription reactions of RNA molecules, the resulting cDNA molecules can be barcoded with a molecular barcode and a carrier barcode and amplified by one or more PCR reactions, such as the first and/or second PCR reactions. A primer pair or multiple primer pairs may be used in the first and/or second PCR reaction. A plurality of forward/reverse primers and a reverse primer may be used in the first and/or second PCR reaction. A plurality of forward/reverse primers and a forward primer may be used in the first and/or second PCR reaction. The first and/or second primer of the plurality of forward/reverse primers may be a forward/reverse primer containing a region complementary to cDNA molecules or barcoded cDNA molecules. The first and/or second primer of the plurality of forward/reverse primers may be a forward/reverse primer containing a region complementary to barcoded cDNA molecules.

[0283] In some embodiments, the forward/reverse primer set comprises one or more forward/reverse primers, where each of the forward/reverse primers of the forward/reverse primer set contains a region complementary to one or more regions upstream or downstream of V-segment cDNA or barcoded cDNA. For example, a plurality of forward/reverse primers comprise a forward/reverse primer containing a region complementary to a region upstream or downstream of a V-segment cDNA or barcoded cDNA, and one or more other forward/reverse primers containing a region complementary to one or more other regions upstream or downstream of a V-segment cDNA or barcoded cDNA. For example, a plurality of forward/reverse primers comprises first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream from the V-segment of cDNA or barcoded cDNA and a second forward/reverse a primer containing a region complementary to the second region upstream or downstream of the V-segment of cDNA or barcoded cDNA. For example, a plurality of forward/reverse primers comprises first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream from the V-segment of cDNA or barcoded cDNA, the second forward/reverse a primer containing a region complementary to the second region upstream or downstream of the V-segment of cDNA or barcoded cDNA, and a third forward/reverse primer containing a region complementary to the third region upstream or downstream of the V-segment cDNA or barcoded cDNA, etc. Primers in a variety of forward/reverse primers can be used to anneal in all possible regions upstream or downstream from all V segments expressed by cells such as immune B cells or T cells, in the sample.

[0284] In some embodiments, the forward/reverse primer set comprises one or more forward/reverse primers, wherein each of the forward/reverse primers of the forward/reverse primer set comprises a region complementary to one or more regions upstream or downstream from C-segment cDNA or barcoded cDNA. For example, a plurality of forward/reverse primers comprise a forward/reverse primer containing a region complementary to regions upstream or downstream of the cDNA C-segment or barcoded cDNA, and one or more other forward/reverse primers containing a region complementary to one or more other regions upstream or downstream of the C-segment of the cDNA or barcoded cDNA. For example, a plurality of forward/reverse primers comprise first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream of the cDNA C-segment or barcoded cDNA and a second forward/reverse a primer containing a region complementary to the second region upstream or downstream of the cDNA C-segment or barcoded cDNA. For example, a plurality of forward/reverse primers contains a first and/or second forward/reverse primer containing a region complementary to the first and/or second region upstream or downstream of the cDNA C-segment or barcoded cDNA, a second forward/reverse a primer containing a region complementary to the second region upstream or downstream of the cDNA C-segment or barcoded cDNA and a third forward/reverse primer containing a region complementary to the third region upstream or downstream of the cDNA C-segment or barcoded cDNA, etc. The primers in the forward/reverse primer array can be used to anneal all possible regions upstream or downstream of all C segments expressed by cells such as immune B cells or T cells. cells in the sample.

[0285] In some embodiments, the plurality of forward/reverse primers comprises one or more forward/reverse primers, wherein each of the forward/reverse primers of the plurality of forward/reverse primers contains a region complementary to one or more regions upstream or downstream of molecular barcode barcoded cDNA. For example, a plurality of forward/reverse primers comprise a forward/reverse primer containing a region complementary to regions upstream or downstream of the molecular barcode of the barcoded cDNA, and one or more other forward/reverse primers containing a region complementary to one or more other regions upstream or downstream of the molecular barcode of the barcoded cDNA. For example, a plurality of forward/reverse primers contains first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream of the molecular barcode of the barcoded cDNA and the second forward/ a reverse primer containing a region complementary to the second region upstream or downstream of the molecular barcode of the barcoded cDNA. For example, a set of forward/reverse primers contains the first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream of the molecular barcode of the barcoded cDNA, the second forward/reverse primer containing a region complementary to the second region upstream or downstream of the molecular barcode of the barcoded cDNA, and a third forward/reverse primer containing a region complementary to the third region upstream or downstream of the molecular barcode barcoded cDNA code, etc. A plurality of forward/reverse primers can be used to anneal all possible regions upstream or downstream of all molecular barcodes expressed by cells, such as immune B cells or T cells, in a sample.

[0286] In some embodiments, the plurality of forward/reverse primers comprises one or more forward/reverse primers, wherein each of the plurality of forward/reverse primers of the plurality of forward/reverse primers contains a region complementary to one or more regions upstream or downstream of transcription in relation to the barcode carrier barcoded cDNA. For example, a plurality of forward/reverse primers comprise a forward/reverse primer containing a region complementary to a region upstream or downstream of the barcode of the barcoded cDNA carrier, and one or more other forward/reverse primers containing a region complementary to one or more other regions upstream or downstream of the barcode carrier of the barcoded cDNA. For example, a plurality of forward/reverse primers contains first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream of the barcode of the barcoded cDNA carrier and the second forward/ a primer reverse channel containing a region complementary to the second region upstream or downstream of the barcode of the barcoded cDNA carrier. For example, a set of forward/reverse primers contains the first and/or second forward/reverse primers containing a region complementary to the first and/or second region upstream or downstream of the barcode of the barcoded cDNA carrier, the second forward/ a reverse primer containing a region complementary to the second region upstream or downstream of the barcode of the barcoded cDNA carrier, and a third forward/reverse primer containing a region complementary to the third region upstream or downstream of the barcode - carrier code of barcoded cDNA, etc. The primers in a plurality of forward/reverse primers can be used to anneal in all possible regions upstream or downstream of all carrier barcodes expressed by cells, such as immune B cells or T cells, in the sample.

[0287] The forward/reverse primers of the plurality of forward/reverse primers further comprise a region that is not complementary to the RNA region. In some embodiments, the region that is not complementary to the RNA region is 5' to the forward/reverse primer region that is complementary to the RNA (i.e., upstream or downstream of the V segment). In some embodiments, the region that is not complementary to the RNA region is located at the 3' position relative to the complementary RNA forward/reverse primer region. In some embodiments, the region that is not complementary to the RNA region is the 5' overhang region. In some embodiments, a region that is not complementary to an RNA region contains a priming site for amplification and/or a second sequencing reaction. In some embodiments, the region that is not complementary to the RNA region contains a priming site for amplification and/or a third sequencing reaction. In some embodiments, the region that is not complementary to the RNA region contains a priming site for the second and third sequencing reactions. In some embodiments, the priming site sequences for the second and third sequencing reactions are identical. Using one or more forward/reverse primers and a reverse primer as described herein, cDNA molecules are amplified using suitable reagents known in the art. In some embodiments, the region is a complementary region of an RNA, such as a constant region or an mRNA polyA tail.

5. Barcodes

[0288] The barcode may be a molecular barcode or a carrier barcode. In some embodiments, a barcode, such as a molecular barcode or a carrier barcode, each may have a length in the range of 2 to 36 nucleotides, 4 to 36 nucleotides, or 6 to 30 nucleotides, or 8 to 20 nucleotides. . 2 to 20 nucleotides, 4 to 20 nucleotides, or 6 to 20 nucleotides. In certain aspects, the melting points of the sample barcodes are within 10°C of each other, within 5°C of each other, or within 2°C of each other. In certain aspects of the barcode melting temperatures, the samples are not within 10°C of each other, within 5°C of each other, or within 2°C of each other. In other aspects, the barcodes are members of the minimal cross hybridization group. For example, the nucleotide sequence of each member of such a set may be substantially different from that of any other member of the set such that no member can form a stable duplex with the complement of any other member under stringent hybridization conditions. In some embodiments, the nucleotide sequence of each member of the minimally cross hybridization group differs from the sequence of each other member by at least two nucleotides. Barcode technologies are described in Winzeler et al. (1999) Science 285:901; Brenner (2000) Genome Biol. 1:1 Kumar et al. (2001) Nature Rev. 2:302; Giaever et al. (2004) Proc. Natl. Acad. sci. USA 101:793; Eason et al. (2004) Proc. Natl. Acad. sci. USA 101:1 1046; and Brenner (2004) Genome Biol. 5:240.

[0289] As used herein, a molecular barcode contains information that is unique to a single molecule from one cell or from one carrier, or for two or more molecules from a plurality or library of molecules from two or more single cells or from two or more single carriers. As used herein, the carrier barcode contains information that is unique for polynucleotides from one cell or from one carrier compared to polynucleotides from another single cell or from another single carrier. In some embodiments, the implementation of the unique information contains a unique sequence of nucleotides. For example, the sequence of a molecular or carrier barcode can be determined when the identity and order of a unique or random nucleotide sequence containing a molecular or carrier barcode is determined. In some embodiments, the first adapter includes a media barcode sequence.

[0290] In some embodiments, the unique information cannot be used to identify the sequence of the target polynucleotide. For example, a molecular barcode can be attached to one target polynucleotide, but the molecular barcode cannot be used to determine the target polynucleotide to which it is attached. In some embodiments, the unique information is not a known sequence linked to the sequence identity of the target polynucleotide. For example, a carrier barcode may be attached to one or more target polynucleotides, but a carrier barcode cannot be used to determine which one or more target polynucleotides it is attached to. In some embodiments, the implementation of the unique information contains a random sequence of nucleotides. In some embodiments, the implementation of the unique information contains one or more unique nucleotide sequences belonging to the polynucleotide. In some embodiments, the implementation of the unique information contains a degenerate nucleotide sequence or a degenerate barcode. A degenerate barcode may contain a composition of variable nucleotide bases or a sequence. For example, a degenerate barcode may be a random sequence. In some embodiments, the sequence complementary to the molecular or carrier barcode is also a molecular or carrier barcode sequence.

[0291] The molecular or carrier barcode can be any nucleotide length. For example, a molecular or carrier barcode may contain at least about 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides. For example, a molecular or carrier barcode can contain at most 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides. In some embodiments, the molecular or carrier barcode has a specific length. For example, a molecular or carrier barcode may be about 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides in length.

[0292] In some embodiments, each molecular or carrier barcode of a plurality of molecular or carrier barcodes has at least about 2 nucleotides. For example, each molecular or carrier barcode of a plurality of molecular or carrier barcodes may be at least about 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides in length. In some embodiments, each molecular or carrier barcode of a plurality of molecular or carrier barcodes has at most about 1000 nucleotides. For example, each molecular or carrier barcode of a plurality of molecular or carrier barcodes may be at most about 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides in length. In some embodiments, each molecular or carrier barcode of a plurality of molecular or carrier barcodes is the same number of nucleotides in length. For example, each molecular or carrier barcode of a plurality of molecular or carrier barcodes may be 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides in length. In some embodiments, one or more molecular or carrier barcodes of a plurality of molecular or carrier barcodes have a different number of nucleotides in length. For example, one or more of the first molecular or carrier barcodes of a plurality of molecular or carrier barcodes may have about or at least about 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides and one or more second molecular or carrier barcodes of the plurality of molecular or carrier barcodes may have about 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, 60, 70, 80, 90, 100, 200, 500 or 1000 nucleotides, where the number of nucleotides of one or more of the first molecular barcodes or carrier barcodes differs from one or more second molecular pcs rih-codes or barcodes of the carrier.

[0293] The number of molecular barcodes may exceed the total number of molecules to be labeled in a plurality of carriers. The number of carrier barcodes may exceed the total number of molecules to be labeled in the plurality of carriers. For example, the number of molecular or carrier barcodes may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70 , 80, 90 or 100 times more than the total number of molecules to be labeled in multiple carriers.

[0294] The number of different molecular barcodes may exceed the total number of molecules that must be labeled in a plurality of carriers. In some embodiments, the number of different molecular barcodes may be at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the total number of molecules to be labeled in the plurality of carriers.

[0295] The number of different molecular barcodes in one carrier may exceed the number of different molecules that must be labeled in one carrier. In some embodiments, the number of different molecular barcodes in one carrier may be at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times the number of different molecules to be labeled in one carrier.

[0296] The number of different carrier barcodes may be less than the total number of molecules to be labeled across multiple carriers. In some embodiments, the number of different media barcodes may be at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 , 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times less than the total number of molecules to be labeled in the plurality of carriers.

[0297] The number of molecules of the amplified product of the carrier barcode polynucleotide molecule in one carrier may exceed the number of different molecules to be labeled in one carrier. In some embodiments, the number of molecules of the amplified product of the carrier-barcoded polynucleotide molecule in one carrier may be at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 , 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more than the number of different molecules to be labeled in one carrier.

[0298] The number of polynucleotide molecules with a carrier barcode in one carrier may be less than the number of different molecules that must be labeled in one carrier. In some embodiments, the number of carrier-barcoded polynucleotide molecules in one carrier may be at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20. 30, 40, 50, 60, 70, 80, 90 or 100 times less than the number of different molecules that must be labeled in one carrier.

[0299] The number of polynucleotide molecules in one carrier with a carrier barcode may be one molecule. The number of non-amplified polynucleotide molecules with a carrier barcode in one carrier may be one molecule.

[0300] In some embodiments, at least about 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% different molecular strokes -codes have the same concentration. In some embodiments, at least about 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 different media barcodes share the same concentration.

[0301] In some embodiments, at least about 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% different molecular strokes -codes have different concentrations. In some embodiments, at least about 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 different media barcodes have different concentration.

[0302] Molecular or carrier barcodes in a population of molecular or carrier barcodes may have at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more different sequences. For example, molecular or carrier barcodes in a population may have at least 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000 or more different sequences. Thus, a plurality of molecular or carrier barcodes can be used to generate at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200 , 300, 400, 500, 600, 700, 800, 900, 1000 or more different sequences from one or more polynucleotides, such as target polynucleotides. For example, a plurality of molecular or carrier barcodes can be used to generate at least 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, L × L0 ⁶ , 2 × L0 ⁶ , 3 × L0 ⁶ , 4 × L0 ⁶ , 5×, 6×l0 ⁶ , 7×l0 ⁶ , 8×l0 ⁶ , 9×l0 ⁶ , l×l0 ⁷ , 2×l0 7 , 3×l0 ⁷ , 4×l0 ⁷ , 5×l0 ⁷ , ⁶ × l0 ⁷ , 7×l0 ⁷ , 8×l0 ⁷ , 9×l0 ⁷ , l×l0 ⁸ , 2×l0 ⁸ , 3×l0 ⁸ , 4×l0 8 , 5×l0 ⁸ , 6×l0 ⁸ , ⁷ × l0 ⁸ , 8×l0 ⁸ , 9×l0 ⁸ , l×l0 ⁹ , 2×l0 ⁹ , 3×l0 ⁹ , 4×l0 ⁹ , 5×l0 9 , 6×l0 ⁹ , 7×l0 ⁹ , ⁸ × l0 ⁹ , 9×l0 ⁹ , l×l0 ¹⁰ , 2×l0 ¹⁰ , 3×l0 ¹⁰ , 4×l0 ¹⁰ , 5×l0 ¹⁰ , 6×l0 ¹⁰ , 7×l0 ¹⁰ , 8×l0 ¹⁰ , 9× l0 ¹⁰ , l×l0 ¹¹ , 2×l0 ¹¹ , 3×l0 ¹¹ , 4×l0 ¹¹ , 5×l0 ¹¹ , 6×l0 ¹¹ , 7×l0 ¹¹ , 8×l0 ¹¹ , 9×l0 ¹¹ , l× l0 ¹² , 2×l0 ¹² , 3×l0 ^{12 ,} 4×l0 ¹² , 5×l0 ¹² , 6×l0 ¹² , 7×l0 ¹² , 8×l0 ¹² , 9×10 ¹² or more different sequences based on one or several polynuclei eotides, such as target polynucleotides. For example, a plurality of molecular or carrier barcodes may be used to generate at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20,000, 25000, 30000, 35000, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, ⁹⁰⁰⁰⁰⁰ , l × l0 ⁶ , 2 × l0 ⁶ , 3 × l0 ^{6 ,} , 6×l0 ⁶ , 7×l0 ⁶ , 8×l0 ⁶ , 9×l0 ⁶ , l×l0 ⁷ , 2×l0 ⁷ , 3×l0 ⁷ , 4×l0 ⁷ , 5×l0 ⁷ , 6×l0 ⁷ , 7×l0 ⁷ , 8×l0 ⁷ , 9×l0 ⁷ , l×l0 ⁸ , 2×l0 ⁸ , 3×l0 ⁸ , 4×l0 ⁸ , 5×l0 8 , 6× ^{l0 8 ,} 7×l0 ⁸ , 8×l0 ⁸ , 9×l0 ⁸ , l×l0 ⁹ , 2×l0 ⁹ , 3×l0 ⁹ , 4×l0 ⁹ , 5×l0 9 , 6×l0 ⁹ , 7×l0 ^{9 ,} 8×l0 ⁹ , 9×l0 ⁹ , l×l0 ¹⁰ , 2×l0 ¹⁰ , 3×l0 ¹⁰ , 4×l0 ¹⁰ , 5×l0 ¹⁰ , 6×l0 ¹⁰ , 7×l0 ¹⁰ , 8×l0 ¹⁰ , 9×l0 ¹⁰ , l×l0 ¹¹ , 2×l0 ¹¹ , 3×l0 ¹¹ , 4×l0 ¹¹ , 5×l0 ¹¹ , 6×l0 ¹¹ , 7×l0 ¹¹ , 8×l0 ¹¹ , 9×l0 ¹¹ , l×l0 ¹² , 2×l0 ¹² , 3×l0 ^12, 4×l0 ¹² , 5×l0 ¹² , 6×l0 ¹² , 7×l0 ¹² , 8×l0 ¹² , 9×10 ¹² or more different sequences of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15000, 20,000, 25000, 30000, 35000, 40000 45000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, L × L0 ⁶ , 2 × L0 ⁶ , 3 × L0 ⁶ , 4 × L0 ⁶ , 5×l0 ⁶ , 6×l0 ⁶ , 7×l0 ⁶ , 8×l0 ⁶ , 9×l0 ⁶ , l×l0 7 , 2×l0 ⁷ , 3×l0 ⁷ , 4×l0 ⁷ , 5× ^{l0 7} , 6×l0 ⁷ , 7×l0 ⁷ , 8×l0 ⁷ , 9×l0 ⁷ , l×l0 ⁸ , 2×l0 ⁸ , 3×l0 ⁸ , 4×l0 ⁸ , 5×l0 ⁸ , 6×l0 ⁸ , 7×l0 ⁸ , 8×l0 ⁸ , 9×l0 ⁸ , l×l0 ⁹ , 2×l0 ⁹ , 3×l0 ⁹ , 4×l0 ⁹ , 5×l0 9 , 6×l0 ^{9 ,} 7×l0 ⁹ , 8×l0 ⁹ , 9×l0 ⁹ , l×l0 ¹⁰ , 2×l0 ¹⁰ , 3×l0 ¹⁰ , 4×l0 ¹⁰ , 5×l0 ¹⁰ , 6×l0 ¹⁰ , 7×l0 ¹⁰ , 8×l0 ¹⁰ , 9×l0 ¹⁰ , l×l0 ¹¹ , 2×l0 ¹¹ , 3×l0 ¹¹ , 4×l0 ¹¹ , 5×l0 ¹¹ , 6×l0 ¹¹ , 7×l0 ¹¹ , 8×l0 ¹¹ , 9×l0 ¹¹ , l×l0 ¹² , 2×l0 ¹² , 3×l0 ^{12 ,} 4×l0 ¹² , 5×l0 ¹² , 6×l0 ¹² , 7×l0 ¹² , 8×l0 ^12.9 ×10 ¹² or more target polynucleotides.

[0303] In some embodiments, one or more molecular barcodes are used to group or sample sequences. In some embodiments, one or more molecular barcodes are used to group or generate samples of sequences, where the sequences in each sample contain the same molecular barcode. In some embodiments, one or more molecular or carrier barcodes are used to group or generate samples of sequences, where the sequences in each sample contain a set of amplicons. In some embodiments, one or more molecular barcodes are used to group or form sequence samples, where the sequences in each sample contain a plurality of sequences, in which the polynucleotides from which the plurality of sequences were derived come from the same polynucleotide molecule in the reaction amplification.

[0304] In some embodiments, one or more carrier barcodes are used to group or sample sequences. In some embodiments, one or more media barcodes are used to group or sample sequences, with the sequences in each sample containing the same media barcode. In some embodiments, one or more carrier barcodes are used to group or generate samples of sequences, with the sequences in each sample containing one or more sets of amplicons. In some embodiments, one or more carrier barcodes are used to group or form sequence samples, where the sequences in each sample contain a plurality of sequences, in which the polynucleotides from which the plurality of sequences were derived come from polynucleotides from a single carrier or a single cell.

[0305] In some embodiments, one or more molecular and carrier barcodes are used to group or sample sequences. In some embodiments, one or more molecular barcodes and carrier barcodes are used to group or generate samples of sequences, with the sequences in each sample containing the same molecular barcode and the same carrier barcode. In some embodiments, one or more molecular and carrier barcodes are used to group or generate samples of sequences, where the sequences in each sample contain one or more sets of amplicons. In some embodiments, one or more molecular barcodes and carrier barcodes are used to group or form sequence samples, where the sequences in each sample contain a plurality of sequences in which the polynucleotides from which the plurality of sequences were derived are derived from the same the same polynucleotide in the amplification reaction and from the same cell or carrier. In some embodiments, one or more molecular and carrier barcodes are not used to combine sequences.

[0306] In some embodiments, one or more molecular barcodes are not used to combine sequences. In some embodiments, one or more molecular barcodes are used to combine sequences. In some embodiments, one or more molecular barcodes are used to group or sample sequences, and a specific target region is used to combine sequences. In some embodiments, one or more carrier barcodes are not used to merge sequences. In some embodiments, one or more carrier barcodes are used to combine sequences. In some embodiments, one or more media barcodes are used to group or sample sequences, and a specific target region is used to combine sequences. In some embodiments, one or more molecular and carrier barcodes are used to combine sequences. In some embodiments, one or more molecular and carrier barcodes are used to group or sample sequences, and a specific target region is used to combine sequences.

[0307] In some embodiments, the combined sequences contain the same molecular barcode. In some embodiments, the combined sequences contain the same carrier barcode. In some embodiments, the combined sequences contain the same molecular barcode and carrier barcode. In some embodiments, one or more molecular or carrier barcodes are used to combine sequences, where the combined sequences contain two or more sequences from a set of amplicons. In some embodiments, one or more molecular or carrier barcodes are used to combine sequences, where the combined sequences contain a plurality of sequences from which polynucleotides were derived from the same polynucleotide molecule in an amplification reaction. In some embodiments, one or more molecular or carrier barcodes are used to combine sequences, where the combined sequences comprise a plurality of sequences in which the polynucleotides from which the plurality of sequences were derived originate from the same cell or carrier.

C. Adapter Ligation

[0308] Prior to ligation of the adapter, the double-barcoded polynucleotides or amplicons may be purified and/or sized. The size of the double-barcoded polynucleotides can be selected to optimize the chosen sequencing method. The desired polynucleotide size is determined by the limitations of the sequencing tools and the purpose for which the sequencing is actually performed. In some embodiments, the desired polynucleotide size is from 0 base pairs (bp) to 100,000 bp. (100 kilobases (kb)), from 50 b.p. up to 50 kb, from 100 bp to 25 kb. In some embodiments, a short sequence sequencer is used to determine the sequence of the polynucleotides generated herein. Typically, optimal polynucleotide sizes for short sequence readers range in length from about 20 base pairs (bp) to 2000 bp, 50 to 1500 bp, 50 to 1250 bp ., from 50 to 1000 bp, from 50 to 750 bp, 50 from 500 to 500 bp, from 100 to 1500 bp, from 100 to 1250 bp, from 100 up to 1000 bp, from 100 to 750 bp, from 100 to 500 bp, from 200 to 1500 bp, from 200 to 1250 bp 1000 bp, 200 bp up to 750 bp or from 250 bp up to 500 bp

[0309] In some embodiments, a long sequence read sequencer is used to determine the sequence of the polynucleotides generated herein. Generally, optimal polynucleotide sizes for short sequence read sequencers range in length from about 1 kilobase (kb) to 100 kb, e.g., 1 kb to 50 kb, 5 kb to 25 kb, 5 kb to 20 kb, or approximately 1 kb, 5 kb, 10 kb, 15 kb or 20 kb.

[0310] To create a collection of polynucleotides of a desired size, a collection of polynucleotides can be obtained with a desired size by changing the conditions of reverse transcription reactions or primer extension, such as modifying the reaction time in the extension step. In some embodiments, a collection of polynucleotides can be fragmented or reduced to the desired length by physical methods (i.e., acoustic shearing and sonication) or enzymatic methods (i.e., using mixtures of non-specific endonucleases and tagging resection using transposase). Polynucleotides of the desired size can be isolated by agarose gel electrophoresis, such as denaturing gel electrophoresis, size exclusion methods, or automated methods, or using commercial kits (Quail et al., Electrophoresis (2012) 33 (23): 3521-3528; Duhaime et al., Environ Microbiol (2012) 14 (9): 2526-2537).

[0311] In some embodiments, double-stranded, double-barcoded polynucleotides are purified prior to selection of appropriately sized polynucleotides, such as by affinity purification. In some embodiments, double-stranded, double-barcoded polynucleotides are denatured prior to selection of appropriately sized polynucleotides. In some embodiments, double-stranded, double-barcoded polynucleotides are denatured by breaking hydrogen bonds between complementary DNA strands. In some embodiments, double-stranded DNA is denatured by the use of an acid or base, a concentrated inorganic solvent, an organic solvent (eg, alcohol or chloroform), radiation, or heat. In some embodiments, double-stranded DNA is denatured by exposure to chemicals such as formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMS), propylene glycol, urea, or NaOH. In some embodiments, double-stranded DNA molecules are treated with NaOH, such as 0.1 M NaOH, to produce single-stranded molecules.

[0312] After sizing and/or purification, a second adapter can be added to double-barcoded adapter-labeled polynucleotides representing polynucleotides produced by a method using the first adapter with a universal priming sequence, a carrier barcode, and a molecular barcode. code. The adapter contains a universal priming sequence that can be used to amplify or sequence double barcodeotides labeled with the adapter. Adapters may contain any known universal priming sequence or fragment thereof. Typical universal priming sequences include P7, C7, P5, or C5 priming sequences.

[0313] Adding a second adapter can be done in any known manner. The adapter can be added to a single stranded polynucleotide or a double stranded polynucleotide. In some embodiments, an adapter is added to a single stranded polynucleotide. In other examples, an adapter, such as a double strand adapter, is added to a double stranded polynucleotide. In some embodiments, a ligase is used to ligate a single stranded adapter. For example, Thermostable App ligase (NEB) or CircLigase II (Epicenter) ligases can be used to ligate a second adapter to a single stranded adapter to a single stranded, adapter-tagged, double-barcoded polynucleotide.

[0314] In some embodiments, a second adapter can be added to single-stranded, adapter-labeled, dual-barcoded polynucleotides by annealing a degenerate splint adapter. For example, a second adapter can be added by adding a duplex hinged (splint) adapter to the end of a single-stranded, labeled adapter, polynucleotide with two bar codes. These splint adapter duplexes contain a paired double-stranded oligonucleotide with a degenerate overhang at one end of the molecule. A degenerate overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases. The degenerate nucleotides in the overhang are annealed to the end of the single-stranded, adapter-labeled, double-barcoded polynucleotide opposite the end of the first adapter sequence. In some embodiments of the method, a 3' ledge splint adapter duplex is annealed at the 3' end of a single-stranded, adapter-labeled, dual-barcoded polynucleotide, at the opposite end to the first adapter. In some embodiments of the method, a 5' ledge splint adapter duplex is annealed at the 5' end of a single-stranded, adapter-labeled, double-barcoded polynucleotide opposite the first adapter. A ligase, such as a blunt-end ligase/TA ligase, can facilitate annealing of a splint adapter duplex with single-stranded, adapter-labeled, dual-barcoded polynucleotides.

[0315] In some embodiments of the method, enzymatic completion of non-template nucleotides can be performed to the end of single-stranded, adapter-labeled, double-barcoded polynucleotides to which the adapter is annealed. In some embodiments, the second adapter is annealed directly to non-template nucleotides in complementary base pairing. In some embodiments, the adapter splint duplex can be annealed with non-template nucleotides to effect the addition of the adapter to the end of the molecule. In some embodiments, an adapter is added to the 3' end of single-stranded, adapter-labeled, double-barcoded polynucleotides. In some embodiments, an adapter is added to the 5' end of single-stranded, adapter-labeled, double-barcoded polynucleotides. A ligase, such as blunt end ligase/TA ligase, can facilitate annealing of a second adapter or a splint adapter duplex with single-stranded, adapter-labeled, double-barcoded polynucleotides.

[0316] The second adapter contains a universal priming sequence or a universal priming site or a contiguous portion of the universal priming sequence or universal priming site sufficient to anneal the complementary sequence. Universal priming sequences or universal priming sites contain oligonucleotide sequences that are complementary to the universal primers or a contiguous portion thereof. Exemplary universal primers are listed in section D below.

D. Amplification and sequencing

[0317] A sample containing double barcoded polynucleotides, with a first and second adapter at or near opposite ends of double barcoded polynucleotides corresponding to one or more target polynucleotide sequences and/or all or part of a cell transcriptome, such as those created in the procedures described above, can be amplified to generate multiple copies of a library of double-barcoded polynucleotides. Amplification can be performed prior to sequencing. In some embodiments, primers with sequencing adapters can be used to amplify one or more sequences in a library. In some embodiments, selected transcripts are amplified and prepared for sequencing.

[0318] In some embodiments, adapter primers with sequencing adapters are used to amplify all transcripts in the library and prepare them for sequencing. In some embodiments, an adapter primer carrying a sequencing adapter and a target specific primer carrying a sequencing adapter are used to amplify a target gene and prepare the target gene for sequencing. In some embodiments, for transcriptome amplification, a cell-specific primer, such as a carrier barcode primer carrying a sequencing adapter, and a primer for an adapter at the opposite (from the carrier barcode with a sequencing adapter) end of the transcript is used to amplify the transcriptome in a selected cell and prepare it for sequencing. Figure 2 depicts an exemplary selected amplification of polynucleotides in a generated library using selected primers as described here.

1. Amplification

[0319] The sample containing the target polynucleotide may contain DNA corresponding to the complete mRNA transcript or fragment(s) thereof that can be amplified. In some cases, the average length of the corresponding mRNA transcript or fragment(s) thereof may be less than about 100, 200, 300, 400, 500, or 800 bp, or less than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides, or less than about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 kilobases. In some cases, a relatively short template target sequence is amplified, such as a carrier containing a template of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bases.

[0320] The amplification reaction may include one or more additives. In some cases, one or more additives are added, such as dimethyl sulfoxide (DMSO), glycerol, betaine (mono) hydrate N,N,N-trimethylglycine = [carboxymethyl]trimethylammonium), trehalose, 7-deaza-2'-deoxyguanosine triphosphate (dC7GTP or 7-deaza-2'-dGTP), BSA (bovine serum albumin), formamide (methanamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (e.g. tetraetamate) (TEA-C1) and tetrapropylammonium chloride (TPrA-Cl), nonionic detergent (e.g. Triton X-100, TWEEN® 20, Nonidet P-40 (NP-40)) or PREXCEL-Q. In some cases, the amplification reaction includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different additives. In other cases, the amplification reaction includes at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different additives.

[0321] Thermal cycling reactions can be carried out on samples contained in reaction volumes (eg, drops). The droplets may be polydisperse, or preferably monodisperse, generated by agitation, sonication, or microfluidics at the T-channel junction, or other methods known to those skilled in the art. Density may exceed 20,000 drops/40 µl (1 nl drops), 200,000 drops/40 µl (100 µl drops). The droplets may remain intact during the thermal cycling process. Drops can remain intact during thermal cycling at densities greater than about 10,000 drops/µl, 100,000 drops/µl, 200,000 drops/µl, 300,000 drops/µl, 400,000 drops/µl, 500,000 drops/µl, 600,000 drops/µl, 700,000 drops /µl, 800000 drops/µl, 900000 drops/µl or 1000000 drops/µl. In other cases, two or more droplets do not coalesce during thermal cycling. In other cases, more than 100 or more than 1000 drops do not coalesce during thermal cycling.

[0322] Any DNA polymerase that catalyzes primer extension can be used, including, but not limited to, E. coli DNA polymerase, E. coli DNA polymerase 1 Klenow fragment, T7 DNA polymerase, T4 DNA polymerase, Taq polymerase , Pfu DNA polymerase, Vent DNA polymerase, bacteriophage 29, REDTaq™, genomic DNA polymerase or sequenase. In some cases, a thermostable DNA polymerase is used. Hot start PCR can also be performed with the reaction heated to 95°C for two minutes before the polymerase is added, or the polymerase can remain inactive until the first heating step in cycle 1. Hot start PCR can be used to minimize non-specific amplification.

[0323] Any number of PCR cycles can be used to amplify DNA, for example, about, more or less than about 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 or 45 cycles. The number of amplification cycles can be about 1-45, 10-45, 20-45, 30-45, 35-45, 10-40, 10-30, 10-25, 10-20, 10-15, 20-35, 25-35, 30-35 or 35-40.

[0324] Amplification of target nucleic acids can be carried out by any known methods. Target nucleic acids can be amplified by polymerase chain reaction (PCR) or isothermal DNA amplification. Examples of PCR methods that can be used include, but are not limited to, quantitative PCR, quantitative fluorescence PCR (QF-PCR), multiplex fluorescence PCR (MF-PCR), real-time PCR (reverse transcription-PCR), single cell PCR, Restriction fragment length polymorphism PCR (PCR-RFLP), PCR-RFLP/reverse transcription-PCR-RFLP, hot-start PCR, nested PCR, in situ colony PCR (polony=Polymerase-colony), in situ ring-array amplification (RCA), digital PCR (dPCR), droplet digital PCR (ddPCR), bridge PCR, picoliter PCR and emulsion PCR. Other suitable amplification methods include ligase chain reaction (LCR), transcriptional amplification, molecular inversion probe PCR (MIP), self-sustained sequence replication, selective amplification of target polynucleotide sequences, cosensnus sequence polymerase chain reaction (CP-PCR), polymerase chain reaction reaction with arbitrary primers (AP-PCR), PCR with degenerate polynucleotide primers (DOP-PCR), and amplification of sequences based on nucleic acids (NABSA). Other amplification methods that can be used here include those described in US patents No. No. 5242774; 5494810; 4988617; and 6,582,938 and also include Q beta replicase mediated RNA amplification. The amplification may be isothermal amplification, such as isothermal linear amplification.

[0325] In some embodiments, amplification is not performed on a solid support. In some embodiments, amplification is not performed on a solid support in a droplet. In some embodiments, the implementation of the amplification occurs on a solid support, when the amplification is not in the droplet.

[0326] The amplification reaction may include one or more additives. In some embodiments, one or more of the additives is dimethyl sulfoxide (DMSO), glycerol, betaine (mono) hydrate (N,N,N-trimethylglycine = [carboxymethyl]trimethylammonium), trehalose, 7-deaza-2'-deoxyguanosine triphosphate (dC7GTP or 7-deaza-2'-dGTP), BSA (bovine serum albumin), formamide (methanamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (e.g. tetraethiammonium chloride (TEA-C1) and tetrapropylammonium chloride (TPA) Cl), nonionic detergent (e.g. Triton X-100, TWEEN® 20, Nonidet P-40 (NP-40)) or PREXCEL-Q. In some embodiments, the implementation of the amplification reaction may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different additives. In other cases, the amplification reaction may contain at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different additives.

[0327] Typically, one or more pairs of primers can be used in the amplification reaction; one primer of a primer pair may be a forward primer and the other primer of a primer pair may be a reverse primer.

[0328] In some cases, the first pair of primers may be used in the amplification reaction; one primer of the first pair may be a forward primer complementary to the sequence of the first target polynucleotide molecule, and one primer of the first pair may be a reverse primer complementary to the second sequence of the first target polynucleotide molecule and the first target locus may be between the first sequence and the second sequence. In some embodiments, the first target locus contains a VH or Vα or Vγ sequence.

[0329] In some cases, a second pair of primers may be used in the amplification reaction; one primer of the second pair may be a forward primer complementary to the first sequence of the second target polynucleotide molecule, and the other primer of the second pair may be a reverse primer complementary to the second sequence of the second target polynucleotide molecule, and the second target locus may be between the first sequence and the second sequence. In some embodiments, the second target locus contains a VL or Vβ or Vδ sequence.

[0330] In some cases, the third pair of primers can be used in the amplification reaction; one primer of the third pair may be a forward primer complementary to the first sequence of the third target polynucleotide molecule, and the other primer of the third pair may be a reverse primer complementary to the second sequence of the third target polynucleotide molecule, and the third target locus may be between the first sequence and the second sequence. In some embodiments, the third target locus contains a barcode, such as a molecular barcode or a carrier barcode.

[0331] In some cases, multiple pairs of primers may be used in the amplification reaction. In some examples, one primer of a primer pair may be a forward primer complementary to a first adapter sequence, and the other primer of a primer pair may be a reverse primer to a second adapter sequence. In some examples, one primer of the primer pair may be a forward primer complementary to the second adapter sequence, and the other primer of the primer pair may be a reverse primer to the first adapter sequence.

[0332] The length of the forward primer and reverse primer may depend on the sequence of the target polynucleotide and the target locus. For example, the length and/or T of the forward primer and reverse primer can be optimized. In some cases, the primer may be more or less than about 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 or 60 nucleotides in length. In some instances, the primer is about 15 to about 20, about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 15 to about 55, about 15 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, about 20 to about 55, or about 20 to about 60 nucleotides in length.

[0333] The primer may be single stranded DNA prior to binding the template polynucleotide. In some cases, the primer initially contains a double-stranded sequence. Suitable primer length may depend on the intended use of the primer, and may vary from about 6 to about 50 nucleotides, or from about 15 to about 35 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. In some embodiments, the primer need not reflect the exact sequence of the template nucleic acid, but may have sufficient complementarity to hybridize to the template. In some cases, the primer may be partially double-stranded prior to binding to the template polynucleotide. The double-stranded primer may have a hairpin loop of about, more than about, or less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 bases. The double-stranded portion of the primer may be about, more than about, less than about, or at least about 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, or 50 bp. The design of suitable primers for amplifying a target sequence is well known in the art.

[0334] Primers may have additional properties that allow detection or immobilization of the primer that do not change the main property of the primer (for example, acting as an initiator of the start point of DNA synthesis). For example, the primers may contain an additional nucleic acid sequence at the 5' end that does not hybridize to the target nucleic acid, but which facilitates cloning or further amplification or sequencing of the amplified product. For example, the additional sequence may contain a primer binding site, such as a universal primer binding site, which may be an adapter. A primer region that is sufficiently complementary to a hybridization template may be referred to herein as a hybridization region.

[0335] Alternatively, the primer used in the methods and compositions described herein may contain one or more universal nucleosides. Non-limiting examples of versatile nucleosides are 5-nitroindole and inosine as described in US Patent Applications Nos. 2009/0325169 and 2010/0167353.

[0336] Primers can be designed according to known parameters to avoid secondary structures and self-hybridization. Different pairs of primers can anneal and melt at approximately the same temperatures, for example, within 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9° C or 10°C from the temperatures of another pair of primers. In some cases, more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000, 5000, 10000 or more primers. Such primers can hybridize to the target polynucleotides described herein.

[0337] Primers can be obtained by various methods, including, but not limited to, cloning of appropriate sequences and direct chemical synthesis using techniques well known in the art (Narang et al., Methods Enzymol. 68:90 ( 1979); Brown et al., Methods Enzymol. 68: 109 (1979)). Primers can also be obtained from commercial sources. The primers may have the same melting point. Primers may have different melting points. Primers can be lengthened or shortened at the 5' end or 3' end to obtain primers with the desired melting point. One of the primers of a primer pair may be longer than the other primer. The 3' anneal length of primers within a pair of primers can vary. In addition, the annealing position of each primer pair can be designed such that the sequence and length of the primer pairs give the desired melting point. The equation for determining the melting point of primers shorter than 25 base pairs is the Wallace rule (T=2 (A+T)+4 (G+C)). Computer programs can also be used to design primers. The TM (melting or annealing temperature) of each primer can be calculated using programs. The primer annealing temperature can be recalculated and increased after any amplification cycle, including but not limited to cycles 1, 2, 3, 4, 5, cycles 6-10, cycles 10-15, cycles 15-20, cycles 20-25, cycles 25-30, cycles 30-35 or cycles 35-40. After the initial rounds of amplification, the 5' portion of the primers can be included in the products of each locus of interest; and thus TM can be recalculated based on both the sequences of the 5' portion of the primers and the 3' portion of each primer.

[0338] Carrying out one or more reactions of the methods described here may include the use of one or more primers. The primer used herein includes a double-stranded, single-stranded, or partially single-stranded polynucleotide that is complementary enough to hybridize to the template polynucleotide. The primer may be single stranded DNA prior to binding to the template polynucleotide. In some embodiments, the implementation of the primer initially contains a double-stranded sequence. The primer site includes the region of the template to which the primer hybridizes. In some embodiments, primers are capable of acting as an initiation point for template-mediated nucleic acid synthesis. For example, primers can initiate template-mediated nucleic acid synthesis when four different nucleotides are present and a polymerization agent or enzyme such as DNA or RNA polymerase or reverse transcriptase.

[0339] The primer pair includes 2 primers: a first primer with a 5' region upstream that hybridizes to the 5' end of the template sequence, and a second primer with a 3' region downstream of the transcription that hybridizes to a region complementary to the 3' end of the template sequence . The primer set includes two or more primers: a first primer or a first set of primers with a 5' region upstream of transcription that hybridizes to the 5' end of a template sequence or a plurality of template sequences, and a second primer or a second set of primers with a 3' region downstream of transcription, which hybridizes to a region complementary to the 3' end of the template sequence or a plurality of template sequences.

[0340] In some embodiments, the primer contains a specific target sequence. In some embodiments, the primer contains a variant barcode sequence. In some embodiments, the primer contains a universal priming sequence. In some embodiments, the primer contains a PCR primer sequence. In some embodiments, the primer contains a PCR primer sequence used to initiate amplification of the polynucleotide. (Dieffenbach, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York (2003)) A universal primer binding site or sequence allows the attachment of a universal primer to a polynucleotide and/or amplicon. Universal primers are well known in the art and include but are not limited to -47F (M13F), alfaMF, AOX3', AOX5', BGHr, CMV-30, CMV-50, CVMf, LACrmt, lambda gt l0F, lambda gt 10R, lambda gt 11F, lambda gt 11R, M13 rev, M13Forward(-20), M13Reverse, male, pQEproseq, pQE, pA-120, pet4, pGAP Forward, pGLRVpr3, pGLpr2R, pKLAC14, pQEFS, pQERS, pucUl, pucU2, reversA, seqIREStam, seqIRESzpet, seqori, seqPCR, seqpIRES-, seqpIRES+, seqpSecTag, seqpSecTag+, seqretro+PSI, SP6, T3-promise, T7-promise, and T7-termInv.

[0341] As used herein, the term "attachment" can refer to both covalent interactions and non-covalent interactions. Attaching a universal primer to a universal primer binding site can be used to amplify, detect and/or sequence a polynucleotide and/or amplicon. The universal primer binding site may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides or base pairs. In another example, the universal primer binding site is at least about 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bp or grounds. In some embodiments, the universal primer binding site contains 1-10, 10-20, 10-30, or 10-100 nucleotides or base pairs. In some embodiments, the universal primer binding site includes about 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 2-90 , 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 1-900, 1-800, 1-700, 1-600, 1 -500, 1-400, 1-300, 1-200, 1-100, 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2-300, 2-200 , 2-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-90, 10-80, 10 -70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-10, 5-900, 5-800, 5-700, 5-600, 5-500, 5-400 , 5-300, 5-200, 5-100, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 25 -900, 25-800, 25-700, 25-600, 25-500, 25-400, 25-300, 25-200, 25-100, 100-1000, 100-900, 100-800, 100-700 100-600 100-500 100-400 100-300 100-200 200-1000 200-900 200-800 -300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600 , 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900 , 600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 nucleotides or base pairs.

[0342] The primers may be of a length compatible with their use in the synthesis of primer extension products. The primer may be a polynucleotide from 8 to 200 nucleotides in length. The length of the primer may depend on the sequence of the template polynucleotide and the locus of the template. For example, the length and/or melting point (TM) of a primer or primer set can be optimized. In some cases, the primer may be about, more than, or less than about 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 or 60 nucleotides in length. In some embodiments, the primers are about 8-100 nucleotides in length, e.g., 10-75, 15-60, 15-40, 18-30, 20-40, 21-50, 22-45, 25-40, 7. - 9, 12-15, 15-20, 15-25, 15-30, 15-45, 15-50, 15-55, 15-60, 20-25, 20-30, 20-35, 20-45, 20-50, 20-55 or 20-60 nucleotides in length and any length in between. In some embodiments, primers are at most about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.

[0343] Typically, one or more pairs of primers can be used in an exponential amplification reaction; one primer of a primer pair may be a forward primer and the other primer of a primer pair may be a reverse primer. In some embodiments, the first primer pair may be used in an exponential amplification reaction; one primer of the first pair may be a forward primer complementary to the sequence of the first template polynucleotide molecule, and the other primer of the first pair may be a reverse primer complementary to the second sequence of the first template polynucleotide molecule, and the first locus of the template may be between the first sequence and the second sequence. In some embodiments, the implementation of the second pair of primers can be used in the amplification reaction; one primer of the second pair may be a forward primer complementary to the first sequence of the second polynucleotide target molecule, and the other primer of the second pair may be a reverse primer complementary to the second sequence of the second polynucleotide target molecule, and the second target locus may be between the first sequence and the second sequence. In some embodiments, the second target locus contains an antibody variable light chain sequence. In some embodiments, a third primer pair may be used in the amplification reaction; one primer of the third pair may be a forward primer complementary to the first sequence of the third template polynucleotide molecule, and the other primer of the third pair may be a reverse primer complementary to the second sequence of the third template polynucleotide molecule, and the third template locus may be between the first sequence and the second sequence.

[0344] One or more primers can anneal to at least a portion of the plurality of template polynucleotides. One or more primers may anneal to the 3' end and/or 5' end of the plurality of template polynucleotides. One or more primers can anneal to the interior of a plurality of template polynucleotides. The interior area may be at least about 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, 100, 150, 200, 210, 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, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides located at the 3' end or 5 ' end of the set of template polynucleotides. One or more primers may contain a fixed panel of primers. One or more primers may contain at least one or more custom primers. One or more primers may contain at least one or more control primers. One or more primers may contain at least one or more primers for housekeeping genes. One or more primers may contain a universal primer. The universal primer can anneal to the universal primer binding site. In some embodiments, one or more custom primers are annealed to the SBC, the target specific region, regions, complementary thereof, or any combination thereof. One or more primers may contain a universal primer. One or more primer primers can be designed to amplify or perform primer extension, reverse transcription, linear extension, non-exponential amplification, exponential amplification, PCR, or any other method for amplifying one or more target polynucleotides or template polynucleotides.

[0345] The target area may contain at least about 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, 100, 150, 200, 220, 230, 240, 250, 260, 270 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 650 700 750 800, 850, 900 or 1000 nucleotides or base pairs. In another example, a specific target region comprises at least about 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bp or grounds; in some embodiments, the target specific region contains about 5-10, 10-15, 10-20, 10-30, 15-30, 10-75, 15-60, 15-40, 18-30, 20-40, 21 -50, 22-45, 25-40, 7-9, 12-15, 15-20, 15-25, 15-30, 15-45, 15-50, 15-55, 15-60 20-25, 20-30, 20-35, 20-45, 20-50, 20-55, 20-60, 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2- 300, 2-200, 2-100, 25-900, 25-800, 25-700, 25-600, 25-500, 25-400, 25-300, 25-200, 25-100 100-1000, 100 -900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600 , 200-500 200-400 200-300 300-1000 300-900 300-800 300-700 300-600 300-500 300-400 400-1000 400-900 -800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700 , 700-1000, 700-900, 700-800, 800-1000, 800-900 or 900-1000 nucleotides or base pairs.

[0346] Primers can be designed according to known parameters to avoid the formation of secondary structures and self-hybridization. In some embodiments, different pairs of primers can anneal and melt at approximately the same temperatures, for example, within 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C. C, 9°C or 10°C melting temperature of another pair of primers. In some embodiments, one or more primers from a plurality of primers may anneal and melt at approximately the same temperatures, such as within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C of the melting point of the other primer. from a variety of primers. In some embodiments, one or more primers from a plurality of primers may anneal and melt at different temperatures than another primer from a plurality of primers.

[0347] The plurality of primers for one or more steps of the methods described herein may comprise a plurality of primers containing about, about at least, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800 , 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20,000, 30000, 40000, 50,000 , 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 50000000, 1000 different primers For example, each primer of the set of primers may contain different regions or sequences specific to the target or template.

[0348] Figure 2 depicts exemplary amplification reaction schemes for amplifying a polynucleotide library provided herein and generated by the methods presented herein. The exemplary methods provided herein use primers associated with a sequencing adapter to be used for sequencing, such as next generation sequencing, to amplify a library for sequencing purposes. Such primers are known and described here. Adapter-labeled sequencing primers are used in the following typical applications.

[0349] In some embodiments, the target gene is amplified for sequencing. In some embodiments, the target gene is amplified using a primer directed to an adapter sequence at one end of the polynucleotide and a target-specific primer positioned to interact with the sequence of the full-length target polypeptide or a selected portion thereof. In some examples, the target sequence may be present in a single cell or multiple cell based library. In some embodiments, one or more target sequences are amplified using primers specific for a universal polynucleotide priming sequence and one or more target specific primers. In some embodiments, two or more target sequences are amplified, each with a universal sequence and target-specific primers, as described. In some embodiments, two or more target sequences are linked, eg, two target sequences that are co-expressed in a cell, eg, target sequences that are co-expressed as a dimer (eg, a heterodimer). Thus, using the provided embodiment, it is possible to obtain paired sequence information such as full length paired sequence information using the provided methods.

[0350] In some embodiments, the entire resulting polynucleotide library can be amplified for sequencing using primers specific for the first adapter universal priming sequence and the second adapter universal priming sequence. Amplification of the polynucleotide libraries provided herein using primers specific for the universal priming sequences located at both ends of the library polynucleotides can yield a transcriptome, or genome, or portion thereof, of all cells used to create the library. Such transcriptome information can be used to obtain data at later time points and/or used to assess the expression (at the transcript level) of several genes in a population of cells included in the sample. In some embodiments, the transcriptomic information of all cells can be analyzed and used to generate clusters of cells with similar transcript expression profiles from the total cell population from which the library was generated.

[0351] In some embodiments, polynucleotides from a single cell can be specifically amplified and sequenced using a primer specific for the carrier barcode sequence and a primer specific for the universal priming site present in the second adapter. In such embodiments, one or more target sequences are amplified as described above and carrier barcode(s) are identified in the target sequence(s) as being of interest. Since all polynucleotides from the same cell are barcoded with the same carrier barcode, this application of the method provides sequence information for all polynucleotides from the selected cell or cells. Amplification of all polynucleotides in the library from the selected cell or cells can then reveal expression profiles or genetic profiles of the cell or cells that express one or more specific target sequences.

2. Sequencing

[0352] After performing one or more of the methods or method steps described herein, the library of resulting polynucleotides can be sequenced.

[0353] Sequencing can be performed by any sequencing method known in the art. In some embodiments, sequencing may be performed at high throughput. Suitable next generation sequencing technologies include the 454 Life Sciences platform (Roche, Branford, CT) (Margulies et al., Nature, 437, 376-380 (2005)); lllumina's Genome Analyzer, GoldenGate Methylation Assay or Infinium Methylation Assays, i.e. Infinium HumanMethylation 27K BeadArray or VeraCode GoldenGate methylation array (Illumina, San Diego, CA; Bibkova et al, Genome Res. 16, 383-393 (2006); and U.S. Patents No. 6,306,597, 7,598,035, 7,232,656), or DNA sequencing by ligation, SOLiD System (Applied Biosystems/Life Technologies; US Pat. or the Helicos True Single Molecule DNA sequencing technology (Harris et al, Science, 320, 106-109 (2008); and U.S. Patents No. 7,037,687, 7,645,596, 7,169,560, and time (SMRTTm), (Soni et al, Clin. Chem. 53, 1996-2001 (2007)). These systems allow multiplex parallel sequencing of many polynucleotides isolated from a sample (Dear, Brief Funct. Genomic Proteomic, 1 (4), 397-416 (2003) and McCaughan et al., J. Pathol, 220, 297-306 (2010) ).

[0354] In some embodiments, polynucleotides are sequenced using dye-modified probe ligation, pyrosequencing, or single molecule sequencing techniques. Polynucleotide sequencing can be performed using sequencing methods such as Helioscope™ single molecule sequencing, Nanopore DNA sequencing, Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS), 454 pyrosequencing, Single Molecule real time sequencing (RNAP), Illumina (Solexa) sequencing, SOLiD sequencing, Ion Torrent™, Ion semiconductor sequencing, Single Molecule SMRT(TM) sequencing, Polony sequencing, DNA nanoball sequencing, and VisiGen Biotechnologies method. Alternatively, sequencing platforms, including but not limited to Genome Analyzer IIx, HiSeq, and MiSeq offered by Illumina's Single Molecule Real Time (SMRTTM) technology, such as the PacBio RS system offered by Pacific Biosciences can be used to sequence polynucleotides. (California) and Solexa Sequencer, True Single Molecule Sequencing (tSMSTM) technology such as the HeliScope™ Sequencer available from Helicos Inc. (Cambridge, Massachusetts). Sequencing may include MiSeq sequencing. Sequencing may include HiSeq sequencing. In some embodiments, polynucleotide sequencing includes paired end sequencing, nanopore sequencing, high throughput sequencing, shotgun sequencing, dye terminator sequencing, multiple primer DNA sequencing, pricer-mediated walking sequencing, dideoxy sequencing. Sanger dideoxy sequencing), Maxim-Gilbert sequencing, pyrosequencing, true single molecule sequencing, or any combination thereof. Alternatively, the polynucleotide sequence can be determined using electron microscopy or using an array of chemically sensitive field-effect transistors (chemFET).

[0355] The method may further include sequencing one or more polynucleotides in the library. The method may further include matching one or more polynucleotide sequences, sequence reads, amplicon sequences, or amplicon array sequences in the library to each other.

[0356] As used herein, "alignment" includes comparing/matching a test sequence, in a format such as a sequence read, with one or more other test sequences, control/reference sequences, or a combination thereof. In some embodiments, "alignment" may be used to determine a consensus sequence from a plurality of sequences or aligned sequences. In some embodiments, the alignment includes determining a consensus sequence from a plurality of sequences, each having an identical molecular or carrier barcode. In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the length of the reference sequence. The actual comparison of two or more sequences can be performed by known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. and Altschul, S., Proc. Natl. Acad. sci. USA, 90-5873-5877 (1993). Such an algorithm is included in the NBLAST and XBLAST programs (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25: 3389-3402 (1997). When using the BLAST and Gapped BLAST programs, any appropriate parameters of the respective programs (eg, NBLAST) may be used. For example, parameters for sequence comparison may be set to score=100, word length=12 (score=100, word length=12) or may vary (eg, W=5 or W=20). Other examples include the Myers and Miller algorithm, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In some embodiments, percent identity between two amino acid sequences can be calculated using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0357] Sequencing may include sequencing at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides or base pairs of polynucleotides. In some embodiments, sequencing includes sequencing at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides or polynucleotide base pairs. In other instances, sequencing includes sequencing at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more nucleotides or polynucleotide base pairs.

[0358] Sequencing may include at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more sequence reads per cycle. As used herein, a sequence read/read includes a nucleotide sequence determined from a sequence or data set obtained using sequencing technologies. In some embodiments, sequencing includes sequencing at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more consecutive reads per cycle. The sequencing may include more than, less than or equal to about 1000000000 sequence reads per cycle. Sequencing may include more than, less than, or exactly about 200,000,000 reads per cycle.

[0359] In some embodiments, the number of sequence reads used to determine the consensus sequence is between about 2 and 1000 sequence reads. For example, the number of sequence reads used to determine the consensus sequence may be approximately 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2-300, 2-200, 2-100 25-900, 25-800, 25-700, 25-600, 25-500, 25-400, 25-300, 25-200, 25-100, 100-1000, 100-900, 100-800, 100 -700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400 , 200-300 300-1000 300-900 300-800 300-700 300-600 300-500 300-400 400-1000 400-900 -600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900 , 700-800, 800-1000, 800-900, or 900-1000 sequence read operations. In some embodiments, the number of reads of the sequences used to determine the consensus sequence is at least about 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 16000, 17000, 18000, 19000, 20,000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90,000, 95000, 100000, 150000, 200000, 250000, 300000, 300000, 300000 350000, 400000, 450000, 500000, 550000, 600000, 650000, 700000, 750000, 800000, 850000, 900000, 950000, 1000000, 50000000 or 1000 reads. In some embodiments, the number of sequence reads used to determine the consensus sequence is at most 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 16000, 17000, 18000, 19000, 20,000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90,000, 95000, 100000, 150000, 200,000, 250000, 300000 350000,000,3500,000 , 400000, 450000, 500000, 550000, 600000, 650000, 700000, 750000, 800000, 850000, 900000, 950000, 1000000, 50000000 or 1000000 read operations

[0360] The method may contain misreads. The method may include determining the number of misreads, for example, to determine reaction conditions or design primer sequences. A comparison of the number of misreads generated under one or more initial reaction conditions or group of conditions can be used to determine a preferred condition or set of conditions. For example, the first method can be performed at a high salt concentration during the PCR reaction, and the second method can be performed at a low salt concentration during the PCR reaction, where the first and second methods are performed essentially the same except for the difference in salt concentration. If the first method results in more misreads, such as more misreads for a particular target polynucleotide sequence or primer, then it can be concluded that lower salt reaction conditions are preferred for that particular target polynucleotide sequence or primer.

II. Cloning and expression of target genes

[0361] In some embodiments, target genes identified and sequenced according to the provided methods can be cloned into vectors for expression in or from cells. You can get a library of expression of target genes, such as immune receptors, such as antibodies or TCR.

[0362] The terms "Antibody Expression Library" or "TCR Expression Library" or "Expression Library" as used herein may refer to a collection of molecules (i.e., two or more molecules) in nucleic acid or protein format. Thus, the term may refer to a collection of expression vectors encoding a plurality of antibody or TCR molecules (i.e., in nucleic acid format), or may refer to a collection of antibody or TCR molecules after they have been expressed in an appropriate expression system (i.e., in nucleic acid format). e. at the protein level). Alternatively, the expression vectors/expression library may be contained in suitable host cells in which they may be expressed. Antibody molecules that are encoded or expressed in the expression libraries of the invention may be in any suitable format, e.g., may be whole antibody molecules or TCRs, or may be fragments of antibodies or TCRs, e.g., single chain antibodies (e.g., scFv antibodies), Fv antibodies , Fab' antibodies, fragments (Fab') ₂ , diabodies, etc. The terms "encoding" and "coding..(name)", in the context of the nucleic acid sequence "encoding/coding ...", or encoding DNA or nucleotide sequence "encoding/encoding" a specific enzyme, as well as other synonymous terms, refers to a DNA sequence that is transcribed and translated into an enzyme when under the control of the appropriate regulatory sequences. A "promoter sequence" is a regulatory region of DNA capable of binding to an RNA polymerase in a cell and initiating transcription downstream (towards the 3' end) of the coding sequence. A promoter is part of a DNA sequence. The sequence region begins with a start codon at the 3' end of the promoter. The promoter sequence includes the minimum number of base elements required to initiate transcription at levels seen above background. However, after RNA polymerase binds to the sequence and transcription is initiated, starting at the start codon (at the 3' end of the promoter), transcription continues downstream in the 3' direction. Within the promoter sequence will be the site of transcription initiation (easily identified by mapping with the SI nuclease), as well as protein-binding domains (consensus sequences) responsible for binding the RNA polymerase.

[0363] Antibody or TCR molecules identified, generated, or selected from, or obtainable from, antibody or TCR expression libraries of the invention represent another further aspect of the invention. Again, these antibodies or TCR molecules can be proteins or nucleic acids encoding antibody or TCR molecules, which in turn can be inserted into a suitable expression vector and/or contained in a suitable host cell.

[0364] The cDNA pool can be subjected to a PCR reaction using polynucleotides that hybridize to the constant region of antibody heavy chain genes and polynucleotides that hybridize to the 5' end of the V _H or Vα or Vγ chain region of antibody or TCR genes. The pool of cDNA can be subjected to a PCR reaction using polynucleotides that hybridize to the constant region of heavy chain genes or antibody alpha or gamma chains or TCRs and polynucleotides that hybridize to the 5' to 5' region of the V _H or Vα or Vγ chain end region. barcoded polynucleotide containing the sequence of the antibody or TCR. The PCR reaction can also be performed to amplify a pool of V _L or Vβ or Vγ chains, such as the kappa and lambda types. The cDNA pool can be subjected to a PCR reaction using polynucleotides that hybridize to the constant region of an antibody light chain gene and polynucleotides that hybridize to the 5' end of the V _L or Vβ or Vγ chain gene region of an antibody or TCR. The cDNA pool can be subjected to a PCR reaction using polynucleotides that hybridize to the constant region of antibody light chain genes and polynucleotides that hybridize to the 5' to 5' end of the V _L or Vβ or Vγ chain gene region of a barcoded polynucleotide containing the sequence antibodies or TCR. Such oligonucleotides or primers may be designed based on known and publicly available information from an immunoglobulin or TCR gene sequence database.

[0365] In some embodiments, the V _H and V _L or Vα and Vβ or Vγ or Vδ sequences can be readily obtained from a library of V _H and VL or Vα and Vβ or Vγ and Vδ sequences obtained by PCR amplification using one or more primers which are non-specific for heavy or light chain genes and in particular for one or both terminal regions of V _H and V _L or Vα and Vβ or Vγ and Vδ polynucleotides. In some embodiments, the V _H and VL sequences can be readily obtained from a library of V _H and V _L or Vα and Vβ or Vγ and Vδ sequences obtained by PCR amplification using primers specific to the carrier barcode region encoded by the polynucleotide . In some embodiments, the V _H and V _L sequences can be readily obtained from a library of V _H and V _L or Vα and Vβ or Vγ and Vδ sequences obtained by PCR amplification using C gene family specific primers or primers , specific for the C-gene. In some embodiments, the V _H and V _L sequences can be readily obtained from a library of V _H and V _L or Vα and Vβ or Vγ and Vδ sequences obtained by PCR amplification using a primer set wherein the first primer is specific to the region of the stroke the carrier code encoded by the polynucleotide and the second primer or plurality of second primers are C gene family specific primers or C gene specific primers. In some embodiments, the V _H and V _L or Vα and Vβ or Vγ and Vδ sequences can be readily obtained from a library of V _H and V _L or Vα and Vβ or Vγ and Vδ sequences obtained by PCR amplification using a primer set, where the first primer is specific to the barcode region of the polynucleotide carrier, and the second primer is specific to the universal sequence.

[0366] In some embodiments, after reverse transcription, the resulting cDNA sequences can be amplified by PCR using one or more primers specific for immunoglobulin genes and, in particular, for one or both of the terminal regions of V _H and V _L or Vα and Vβ or Vγ and Vδ polynucleotides. In some embodiments, the V _H and V _L sequences can be obtained from a library of V _H and V _L or Vα and Vβ or Vγ and Vδ sequences obtained by PCR amplification using V gene family specific primers or V specific primers. -genes (Nicholls et al. J. Immunol. Meth., 1993, 165: 81; WO 93/12227) or designed according to standard methods known in the art, based on available sequence information. (The V _H and V _L or Vα and Vβ or Vγ and Vδ sequences can be ligated, typically to an intermediate spacer sequence (eg, encoding a flexible in-frame peptide spacer) to form a cassette encoding a single chain antibody). V-region sequences can conveniently be cloned as cDNA or PCR amplification products for immunoglobulin-expressing cells. The V _H and V _L or Vα and Vβ or Vγ and Vδ regions are sequenced, optionally using the methods described here and in particular after certain steps have been carried out as noted here (for example, after single cell PCR; after appearance on the surface of mammalian cells or other cells after FACS screening, and so on). Sequencing can be used, among other things, to verify that the degree of diversity is at an acceptable level. Sequencing may include high throughput sequencing, deep sequencing (in which the same gene is sequenced from many individual samples to detect differences in sequences), or combinations thereof.

[0367] In some embodiments, natural combinations of V _H and V _L or Vα and Vβ or Vγ and Vδ need not be physically linked using the methods described herein. In some embodiments, the cDNA, barcoded polynucleotides, or PCR-amplified barcoded cDNAs are not physically linked. In some embodiments, cDNA, barcoded polynucleotides, or PCR-amplified barcoded cDNAs are not physically linked in the same reaction or vehicle.

[0368] In some embodiments, natural combinations of V _H and V _L or Vα and Vβ or Vγ or Vδ are physically linked using, in addition to cDNA primers, a single primer or multiple primers to the 5' end of V _H or Vα or Vγ genes and another primer or primer set to the 5' end of the V _L or Vβ or Vδ gene. These primers also contain complementary tails of an additional sequence to allow self-assembly of the V _H and V _L or Vα and Vβ or Vγ and Vδ genes. After PCR amplification and binding, the likelihood of obtaining mixed products, in other words, mixed variable regions, is minimal, since the amplification and binding reactions were carried out within each cell. The risk of mixing can be further reduced by using larger reagents, such as digoxigenin-labeled nucleotides, to further ensure that cDNA V-region pairs do not leave the cell compartment and are mixed, but still remain in the cell for PCR amplification and binding. The amplified sequences are linked by hybridization of complementary end sequences. After binding, sequences can be isolated from cells for use in further steps of the method described here. For example, isolated DNA can be amplified by PCR using terminal primers, if necessary, and cloned into vectors that are plasmids, phages, cosmids, phagemids, viral vectors, or combinations thereof, as detailed below. Convenient restriction enzyme sites can be included in hybridized sequences to facilitate cloning. These vectors can also be stored as a library of associated variable regions for later use.

[0369] In some embodiments, in which it is desirable to provide additional combinations of V _H and V _L or Vα and Vβ or Vγ and Vδ, an expression system is selected to facilitate the action. For example, bacteriophage expression systems allow for random recombination of heavy and light chain sequences. Other suitable expression systems are also known to those skilled in the art.

[0370] It should be noted that in the case of V _H and V _L or Vα and Vβ or Vγ and Vδ sequences derived from a human, in some embodiments, it may be preferable to obtain genetic chimeras of these sequences with complete human Fc. As used herein, "chimeric production/chimerization" refers to an immunoglobulin or TCR where the heavy and light chain variable regions or the Vα and Vβ or Vγ and Vδ regions are not of human origin and where the heavy and light chain constant regions or the Vα and Vβ or chain Vγ and Vδ come from humans. This is influenced by amplification and cloning of variable domains in human Fc. The human Fc may be part of a vector or a single molecule, or an Fc library may be used. In a preferred embodiment, chimerized molecules grown in mammalian cells, such as CHO cells, are FACS screened twice to enrich the cell population for cells expressing the antibody of interest. Chimerized antibodies or TCRs are characterized either by sequencing followed by functional characterization or by direct functional characterization or kinetics. Growth, screening and characterization are detailed below.

[0371] It is important to note that the PCR reactions described above are described for cloning antibodies in the form of IgG. They are preferred because they are generally associated with a more mature immune response and tend to exhibit higher affinity than IgM antibodies, making them more desirable for certain therapeutic and diagnostic applications. However, it is clear that polynucleotides can be engineered to allow cloning of one or more other forms of immunoglobulin molecules, such as IgM, IgA, IgE, and IgD, if desired or appropriate.

[0372] Once an antibody or TCR has been identified and the appropriate population of said cells has been isolated at the appropriate time and optionally enriched as described above, antibody or TCR expression libraries may not be generated immediately, provided that the genetic material contained in the cells , can be saved in its original state, which allows you to make the library later. Thus, for example, cells, cell lysate, or nucleic acid, such as RNA or DNA derived from them, can be stored to a later date by appropriate methods, such as freezing, and expression libraries can be created at a later date when necessary.

[0373] Once a library of expression vectors has been created, the encoded antibody molecules can then be expressed in a suitable expression system and screened using appropriate techniques that are well known and documented in the art. Thus, the method of the invention described above may include the additional steps of expressing the library of expression vectors in an appropriate expression system and screening the expressed library for antibodies with the desired properties, as explained in more detail below.

[0374] As indicated herein, polynucleotides obtained by the methods of the present invention, including polynucleotides encoding an antibody or TCR sequences, may include, but are not limited to, those encoding the amino acid sequence of an antibody or TCR fragment, themselves, non-coding sequences for the entire antibody or TCR or a portion thereof, coding sequences for the antibody or TCR, a fragment or part thereof, as well as additional sequences, such as coding sequences for at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, along with additional non-coding sequences, including but not limited to 5' and 3' non-coding sequences such as transcribed non-translated sequences that play a role in transcription, mRNA processing, including c splicing and polyadenylation signals (eg, ribosome binding and mRNA stability); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionality. Thus, an antibody coding sequence can be fused to a marker sequence, such as a peptide coding sequence that facilitates purification of the fused antibody or TCR (or purification of a portion or fragment of an antibody or TCR).

[0375] The primary PCR products can then optionally be subjected to a second PCR reaction with new polynucleotide sets that hybridize to the 5' and 3' ends of the antibody or TCR variable domains V _H , V _L kappa and V _L lambda or Vα and v or Vγ and Vδ (depending on whether the primary PCR reaction using the new polynucleotide sets was designed to amplify portions of antibody heavy or light chain genes or TCR genes, Vα or Vβ, or Vγ or Vδ TCR genes). These polynucleotides preferably include DNA sequences specific for a particular set of restriction enzymes (ie, restriction enzyme sites) for subsequent cloning. The restriction enzymes chosen should be chosen so as not to cut human antibodies or TCR V gene segments. Such polynucleotides can be designed based on known and publicly available immunoglobulin or TCR gene sequences and restriction enzyme database information. However, preferred sites for restriction enzymes are Ncol, Hind III, M, and Notl. The products of such secondary PCR reactions are repertoires of different fragments/domains of V-heavy, V-light kappa and V-light lambda antibodies. Therefore, this type of secondary PCR reaction is usually performed when the expression library format of interest is an scFv or Fv format where only the VH and VL or Vα and V or Vγ and Vδ domains of an antibody or TCR are present.

[0376] PCR products can also be subjected to a PCR reaction with novel primer sets that hybridize to the 5' and 3' ends of the barcoded polynucleotides. These polynucleotides may advantageously include DNA sequences specific for a particular set of restriction enzymes (ie, restriction enzyme sites) for subsequent cloning. The restriction enzymes chosen should be chosen so as not to cut within the sequence of the human antibody or TCR V gene segments. Such polynucleotides can be designed based on known and publicly available immunoglobulin or TCR gene sequences and restriction enzyme database information. However, preferred restriction enzyme sites are Ncol, Hind III, Mlul and Notl. The products of such secondary PCR reactions are repertoires of different V _H , V _L kappa and V _L lambda antibody fragments/domains or Vα and Vβ or Vγ and Vδ TCR fragments/domains.

[0377] One of skill in the art will appreciate that Fv or Fab heavy or light chain fragments, Vα or Vβ chain or Vγ or Vδ chain fragments, or single chain antibodies or TCRs can also be used with this system. The heavy or light chain or the Vα or Vβ chain or the Vγ or Vδ chain can be mutagenized, followed by the addition of the complementary chain to the solution. The two chains are then allowed to combine and form a functional antibody fragment. The addition of random non-specific light or heavy chain or Vα or Vβ or Vγ or Vδ chain sequences allows for a combinatorial system to create libraries of various components.

[0378] Libraries of such repertoires of cloned fragments containing regions of the variable heavy chain or Vα chain or Vγ chain or fragments thereof, and/or variable regions of the light chain or Vβ chain or Vδ chain of antibody or TCR genes or fragments thereof derived from B - or T-lymphocytes of immunostimulated hosts, as described in this description, form additional aspects of the invention. Such libraries containing cloned variable regions may optionally be inserted into expression vectors, thereby forming expression libraries.

[0379] In some embodiments, the implementation of the PCR reactions can be organized in such a way as to preserve all or part of the constant regions of various antibodies or TCR chains contained in an isolated population of immune cells. This is desirable when the expression library format is a Fab format, where the heavy or alpha or gamma chain component contains VH or Vα or Vγ and C _H or Cα or Cγ domains and the light chain or Vβ chain or Vγ chain component contains V _L or Vβ or Vδ chain and C _L or β or δ domains. Again, libraries of such cloned fragments comprising all or part of the antibody constant regions or TCR chains are further aspects of the invention.

[0380] These nucleic acids can easily include sequences in addition to the polynucleotide of the present invention. For example, a multi-cloning site containing one or more endonuclease restriction sites can be inserted into a nucleic acid to aid in polynucleotide isolation. In addition, translated sequences may be inserted to aid in the isolation of a translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence can be a useful tool for purifying the proteins of the present invention. The nucleic acid of the present invention, excluding the coding sequence, is optionally a vector, adapter or linker for cloning and/or expression of a polynucleotide of the present invention.

[0381] Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in polynucleotide isolation, or to improve the introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters and linkers is well known in the art. (See, for example, Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. Ed., John Wiley and Sons, Inc.) or J. Sambrook et al., “Molecular Cloning: A Laboratory Manual, ” 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, NY).

[0382] The libraries disclosed in this document can be used in various applications. As described in this document, the library contains many molecules. In some embodiments, the library contains a plurality of polynucleotides. In some embodiments, the library contains a plurality of primers. In some embodiments, the library contains multiple sequential reads of one or more polynucleotides, amplicons, or sets of amplicons. The library can be stored and used multiple times to generate samples for analysis. Some applications include, for example, the genotyping of polymorphisms, the study of RNA processing, and the selection of clonal representatives for sequencing according to the methods presented herein. Libraries can be created containing multiple polynucleotides, such as primers or libraries for sequencing or amplification, where the multiple polynucleotides include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 15000, 2000, 3000 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20,000, 30000, 40000, 50000, 60,000, 70000, 80000, 90000, 100,000 , 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 50000000, 100000000 or more molecular or carrier barcodes. In some embodiments, the polynucleotide libraries contain a plurality of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 , 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 10000 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20,000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300000, 400000, 500000, 600000, 700000, 800000, 800000, 800000 , 900000, 1000000, 50000000, 100000000 or more unique polynucleotides, where each unique polynucleotide contains one or more molecular barcodes and carrier barcodes.

III. Transcriptomic analysis

[0383] In some embodiments, the provided methods can be used to elucidate cell transcriptome information, which can be done in conjunction with capture of a target polynucleotide sequence, such as an antibody or TCR of a particular cell. In some embodiments, a cell or plurality of cells identified by a target polynucleotide sequence may be characterized by their transcriptional state, including with respect to a particular state, trait, or attribute of the cell. In some embodiments, individualized transcriptome profiles of cells can be determined and information related to one or more features of the cell provided. Examples of such traits include, but are not limited to, an activation state, a proliferation state, a depletion state, a transition state, a cell cycle stage, or another parameter associated with the functional or phenotypic state of the cell.

[0384] In some embodiments, transcription state information can be used to identify cells expressing a particular target gene, such as an antibody or TCR gene, that exhibits a desired or simply interesting response. In some aspects, the provided methods allow the transcriptome profile of a cell to be matched to a target polynucleotide, such as an antibody or TCR, amplified or sequenced from a cell. In specific embodiments, the transcriptome information and target polynucleotide sequence are matched by the amplified and sequenced transcriptome and target polynucleotides having the same carrier barcode identifying profiles of the transcriptome and target polynucleotides derived from the same cell.

[0385] Various methods for processing transcriptome data are known in the art. In some aspects, the data obtained using the methods can be visualized on a map. A map of the number and location of sample targets may be constructed using information generated using the methods described herein. The map can be used to determine the physical location of a target. The map can be used to locate multiple targets.

[0386] In some embodiments, the system comprises a computer-readable medium that includes code for providing data parsing to fetch sequence data generated using the provided methods. Examples of data analysis functions that may be provided by data analysis software include, but are not limited to: (i) decoding/demultiplexing algorithms for sample carrier barcode, molecular barcode and target sequence or transcriptome data, derived from the sequencing of the polynucleotide library, (ii) algorithms to determine the number of reads per gene per cell and the number of unique transcriptional molecules per gene per cell from the data and generate summary tables, (iii) statistical analysis of the sequence data, e.g. to cluster cells from the data gene expression or to predict confidence intervals to determine the number of transcript molecules per gene per cell, etc., (iv) algorithms to identify subpopulations of rare cells, e.g. using basic component analysis, hierarchical clustering, k-means clustering, self-organizing maps , neural networks, etc., (v) sequence alignment capabilities to align gene sequence data with known reference/reference sequences and detect mutations, polymorphic markers and splicing variants, and (vi) automatic molecular label clustering to compensate for amplification or sequencing errors.

[0387] In some embodiments, computer programs may be used to assemble the transcriptome. Exemplary computational programs for short read transcriptome assemblies include those described by Robertson et al., Nat Methods. 2010;7:909-12; Grabherr et al., Nat Biotechnol. 2011;29:644-52; Schulz et al., Bioinformatics. 2012;28:1086-92, and Xie et al., Bioinformatics. 2014;30:1660-6. Transcriptome assembly can be challenging due to large variation in transcript expression levels, sequencing bias errors, and alternative splicing. Thus, bundling transcriptomes based on k-mer lengths, or using a fixed k-mer value, can be used to compensate for different degrees of transcriptome abundance and improve transcriptome assembly (see, e.g., Robertson et al., Nat Methods. 2010; 7:909-12, Grabherr et al., Nat Biotechnol. 2011;29:644-52 and Surget-Groba Genome Res. 2010;20:1432-40).

[0388] In some embodiments, commercially available software may be used to perform all or part of the data analysis. In some embodiments, the data analysis software may include options for outputting sequencing results in desired graphical formats, such as "heat maps", which indicate the number of copies of one or more genes found in each cell in a collection of cells. In some embodiments, the data analysis software may further comprise algorithms for extracting biological significance from the sequencing results, for example, by correlating the copy number of one or more genes found in each cell of the cell sample with cell type, rare cell type or cell type, obtained from a subject having a particular disease or condition. In some embodiments, the data analysis software may further comprise algorithms for comparing cell populations in different biological samples.

[0389] In some embodiments, all data analysis functionality may be packaged in a single software package. In some embodiments, a complete set of data analysis capabilities may comprise a set of software packages. In some embodiments, the data analysis software may be a stand-alone package that is provided to users independently of the instrumentation system. In some embodiments, the software may be available online (web-based) and may allow users to exchange data.

[0390] In some examples, cluster analysis can be performed to identify one or more different cell populations. In some examples, cell populations are clustered based on the expression of one or more target genes. The presence, activation or suppression of known genes or transcripts that reflect the state of transcription in a cell can be used to cluster cells of multiple cells.

[0391] In some embodiments, the transcriptome genes of interest are identified by a carrier barcode and matched or combined with the amplification and sequencing yield of the full-length target molecule of interest, such as an antibody or TCR. Accordingly, in the practice of the provided methods, each carrier barcode corresponds to or can be annotated with information about the number of genes (eg, transcriptome) and target molecule (eg, full length antibody or TCR).

[0392] In some embodiments, an expression profile represented by nucleic acid sequences present in the transcriptome can be created. Expression profiles can be generated from a subset of cells, such as cells expressing one or more target genes and sharing a common carrier barcode.

V. Diagnosis

[0393] In some embodiments, the method may further include diagnosing, predicting, monitoring, treating, improving, and/or preventing a disease, disorder, symptom, and/or condition in a subject. In some embodiments, the method may further comprise diagnosing, predicting, monitoring, treating, ameliorating, and/or preventing a disease, disorder, symptom, and/or condition in a subject based on the presence, absence, or specific level of a target polynucleotide and/or a particular transcriptional state in cell, such as the state of the cell described here above. In some embodiments, the method may further comprise diagnosing, predicting, monitoring, treating, improving, and/or preventing a disease, disorder, symptom, and/or condition in a subject based on the presence, absence, or a certain level of one or more target polynucleotides and/or a condition. transcription of one or more cells.

[0394] In some embodiments, the implementation of the method may further include diagnosing, predicting, monitoring, treating, improving and / or preventing a disease, disorder, symptom and / or condition in a subject based on the presence, absence, a certain level or sequence corresponding to one or more sequences obtained using the methods described herein. For example, a diagnosis of a disease can be made based on the presence, absence, specific level, or sequence corresponding to a variant sequence obtained using the methods described herein. In some embodiments, the method may further include diagnosing, predicting, monitoring, treating, ameliorating, and/or preventing a disease, disorder, symptom, and/or condition in a subject based on the presence, absence, level, or sequence of one or more sequence reads obtained. using the methods described in this document. In some embodiments, the method may further comprise diagnosing, predicting, monitoring, treating, improving, and/or preventing a disease, disorder, symptom, and/or condition in a subject based on the presence, absence, a particular level, or sequence corresponding to one or more of the consensus sequences. obtained using the methods described in this document. In some embodiments, the method may further comprise diagnosing, predicting, monitoring, treating, improving, and/or preventing a disease, disorder, symptom, and/or condition in a subject based on determining the level (e.g., amount or concentration) of a target polynucleotide in a sample. The level of a target polynucleotide in a sample can be determined based on one or more sequence reads, sequences, consensus sequences, or any combination thereof. The level of each of the multiple target polynucleotides in a sample can be determined using the methods described herein. The level of each of the multiple target polynucleotides in a sample can be determined based on the number of sequence reads, consensus sequences, or any combination of each of the multiple target polynucleotides. For example, the level of the first target polynucleotide and the level of the second target polynucleotide can be determined using the methods described herein.

[0395] In some embodiments, the first and second target polynucleotides of the plurality of target polynucleotides are identical. For example, the first target polynucleotide may contain a first copy of the mRNA molecule, and the second target polynucleotide may contain a second copy of the mRNA molecule. In some embodiments, the first and second target polynucleotides are different. For example, the first target polynucleotide may comprise a first mRNA molecule and the second target polynucleotide may comprise a second mRNA molecule transcribed from a gene different from that of the first mRNA molecule. For example, the first target polynucleotide may contain the first allele, and the second target polynucleotide may contain the second allele. For example, the first target polynucleotide may contain a wild-type sequence, and the second target polynucleotide may contain another sequence variant.

[0396] In some embodiments, the method may further include diagnosing or predicting a disease, disorder, symptom, and/or condition in a subject with at least a 50% probability. For example, the diagnosis or prognosis of a disease, disorder, symptom, and/or condition in a subject can be determined with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, 95 %, 98%, 99% or 100% probability. In some embodiments, the diagnosis or prognosis of a disease, disorder, symptom and/or condition in a subject can be determined with a probability of 50-100%. For example, the diagnosis or prognosis of a disease, disorder, symptom and/or condition in a subject can be determined with 60%-100%, 70%-100%, 80%-100%, 90%-100%, 50-90%, 50 -80%, 50-70%, 50-60%, 60-90%, 60-80%, 60-70%, 70-90%, 70%-80% or 80%-90% probability.

[0397] In some embodiments, the presence, absence, level, sequence of a target polynucleotide such as a biomarker, or any combination thereof, in a subject can be determined with a probability of at least 50%. For example, the presence, absence, level, target polynucleotide sequence itself, or any combination thereof in a subject can be determined with at least 50%, 55%, 60%, 65%, 70%, 75%, 80% 85%, 90 %, 95%, 98%, 99% or 100% probability. In some embodiments, the presence, absence, level, the target polynucleotide sequence itself, or any combination thereof in a subject can be determined with a probability of 50-100%. For example, the presence, absence, level, target polynucleotide sequence itself, or any combination thereof in a subject can be determined with 60%-100%, 70%-100%, 80%-100%, 90%-100%, 50%- 90%, 50% -80%, 50% -70%, 50% -60%, 60% -90%, 60% -80%, 60% -70%, 70% -90% 70% -80% or 80%-90% chance.

B. Enzymes

[0398] The methods and kits disclosed herein may contain one or more enzymes. Examples of enzymes include, but are not limited to: ligases, reverse transcriptases, polymerases, and restriction nucleases.

[0399] In some embodiments, attachment of an adapter to polynucleotides involves the use of one or more ligases. Examples of ligases include, but are not limited to: DNA ligases such as DNA ligase I, DNA ligase III, DNA ligase IV, T4 DNA ligase, and RNA ligases such as T4 RNA ligase I and T4 RNA ligase II.

[0400] The methods and kits disclosed herein may further include the use of one or more reverse transcriptases. In some embodiments, the reverse transcriptase is HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, and telomerase reverse transcriptase. In some embodiments, the reverse transcriptase is an M-MLV reverse transcriptase.

[0401] In some embodiments, the methods and kits disclosed herein include the use of one or more proteases.

[0402] In some embodiments, the methods and kits disclosed herein include the use of one or more polymerases. Examples of polymerases include, but are not limited to, DNA polymerases and RNA polymerases. In some embodiments, the DNA polymerase is DNA polymerase I, DNA polymerase II, DNA polymerase III holoenzyme, and DNA polymerase IV. Commercially available DNA polymerases include, but are not limited to: Bst 2.0 DNA polymerase, Bst 2.0 WarmStart™ DNA polymerase, Bst DNA polymerase, Sulfolobus DNA polymerase IV, Taq DNA polymerase, 9°NTMm DNA polymerase, Deep VentR™ (exo-) DNA Polymerase, Deep VentR™ DNA Polymerase, Hemo KlenTaq™, LongAmp® Taq DNA Polymerase, OneTaq® DNA Polymerase, Phusion® DNA Polymerase, Q5TM High-Fidelity DNA Polymerase, Therminator™ γ DNA Polymerase, Therminator™ DNA Polymerase, Therminator™ II DNA polymerase, Therminator™ III DNA polymerase, VentR® DNA polymerase, VentR® (exo-) DNA polymerase, Bsu DNA polymerase, phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, Terminal Transferase, Titanium® Taq Polymerase, KAPA Taq DNA polymerase and KAPA Taq Hot Start DNA polymerase.

[0403] In some embodiments, the polymerase is an RNA polymerase, such as RNA polymerase I, RNA polymerase II, RNA polymerase III, E. coli (A) polymerase, phi6 RNA polymerase (RdRP), Poly(U ) polymerase, SP6 RNA polymerase and T7 RNA polymerase.

VI. Kits and additional reagents

[010] This section describes products or kits that contain one or more reagents for implementing the provided methods. Kits may optionally include one or more components, such as instructions for use, devices, and additional reagents (for example, sterile water or saline solutions for diluting and/or reconstituting reagents or components), and components, such as test tubes, containers, and syringes for using methods. In some embodiments, the kits may further contain reagents for sample collection or sample preparation and processing. In some embodiments, kits may be provided as articles that include packaging materials for packaging reagents and kit components. For example, kits may contain containers, bottles, test tubes, vials, and any packaging material suitable for separating or organizing kit components.

[0404] The methods and kits disclosed herein may include the use of one or more reagents. Examples of reagents include, but are not limited to: PCR reagents, ligation reagents, reverse transcription reagents, enzyme reagents, hybridization reagents, sample preparation reagents, affinity capture reagents, solid supports such as beads, and reagents for purifying nucleic acids and/or isolating them.

[0405] The solid support may contain virtually any insoluble or solid material, and a solid support that is water insoluble is often chosen. For example, the solid support may contain or consist essentially of silica gel, glass (e.g., controlled pore glass (CPG)), nylon, Sephadex®, Sepharose®, cellulose, metal surface (e.g., steel, gold, silver, aluminium, silicon and copper), magnetic material, plastic material (for example, polyethylene, polypropylene, polyamide, polyester, polyvinylidene difluoride (PVDF)) and the like. Examples of beads for use in accordance with embodiments may include an affinity moiety that allows the bead to interact with a nucleic acid molecule. The solid phase (eg, bead) may contain a binding pair component (eg, avidin, streptavidin, or a derivative thereof). For example, the bead may be a streptavidin-coated bead, and the nucleic acid molecule may include a biotin group for immobilization on the bead. In some cases, each polynucleotide molecule may include two affinity moieties, such as biotin, to further stabilize the polynucleotide. The beads may have additional properties for immobilizing nucleic acids or for use in subsequent screening or selection processes. For example, the bead may include a binder moiety, a fluorescent label, or a fluorescent quencher. In some cases, the pellet may be magnetized. In some cases, the solid support is a bead. Examples of beads include, but are not limited to: streptavidin beads, agarose beads, magnetic beads, Dynabeads® microbeads, MACS®, antibody-conjugated beads (e.g., anti-immunoglobulin-coated microbeads), protein A-conjugated beads, protein G, protein A/G-conjugated beads, protein L-conjugated beads, polynucleotide-dT conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, antifluorochromic microbeads and BcMag™ with carboxy-terminal magnetic granules. The granules or particles may be swellable (eg polymer granules such as Wang resin) or non-swellable (eg CPG). In some embodiments, the implementation of the solid phase is essentially hydrophilic. In some embodiments, the implementation of the solid phase (eg, granule) is essentially hydrophobic. In some embodiments, the solid phase contains a binding pair component (eg, avidin, streptavidin, or a derivative thereof) and is substantially hydrophobic or substantially hydrophilic. In some embodiments, the solid phase contains a binding couple element (eg, avidin, streptavidin, or a derivative thereof) and has a binding capacity greater than about 1350 picomoles of free entrapment agent (eg, free biotin) per mg of solid support. In some embodiments, the binding capacity of the solid phase containing the binding pair element is greater than 800, 900, 1000, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1800, 2000 picomoles of free capture agent per mg of solid support . Other examples of granules suitable for use in the invention are gold colloids or granules such as polystyrene or quartz granules. As such, granules of any radius can be used. Examples of granules may include granules having a radius in the range of 150 nm to 10 µm. Other sizes may also apply.

[0406] The methods and kits disclosed herein may include the use of one or more buffers. Examples of buffers include, but are not limited to, wash buffers, ligation buffers, hybridization buffers, amplification buffers, and reverse transcription buffers. In some embodiments, the hybridization buffer is a commercially available buffer such as TMAC Hyb solution, SSPE hybridization solution, and ECONOTM hybridization buffer. The buffers disclosed herein may contain one or more detergents.

[0407] The methods and kits disclosed herein may include the use of one or more carriers. Carriers may enhance or improve the performance of one or more of the reactions described herein (eg, ligation, reverse transcription, amplification, hybridization reactions). Carriers can reduce or prevent non-specific loss of molecules or any of their products (eg, polynucleotides and/or amplicons). For example, the carrier can reduce non-specific loss of the polynucleotide due to surface absorption. The carrier may reduce the affinity of the polynucleotide to a surface or substrate (eg container, Eppendorf tube, pipette tip). Alternatively, the carrier may increase the affinity of the polynucleotide for a surface or substrate (eg, bead, mesh, glass, slide, or microarray). Carriers can protect the polynucleotide from degradation. For example, carriers can protect the RNA molecule from ribonucleases. Alternatively, the carriers may protect the DNA molecule from DNase. Examples of carriers include, but are not limited to, polynucleotides such as DNA and/or RNA, or polypeptides. Examples of DNA carriers include plasmids, vectors, polyadenylated DNA, and DNA polynucleotides. Examples of RNA carriers include polyadenylated RNA, phage RNA, MS2 phage RNA, E. coli RNA, yeast RNA, yeast tRNA, mammalian RNA, mammalian tRNA, short polyadenylated synthetic ribonucleotides, and RNA polynucleotides. The carrier RNA may be polyadenylated RNA. Alternatively, the carrier RNA may be non-polyadenylated RNA. In some embodiments, the carrier is derived from a bacterium, yeast, or virus. For example, the carrier may be a polynucleotide or polypeptide derived from a bacterium, yeast, or virus. For example, the carrier is a Bacillus subtilis protein. In another example, the carrier is an Escherichia coli polynucleotide. Alternatively, the carrier is a mammalian (eg, human, mouse, goat, rat, bovine, sheep, pig, dog, or rabbit), avian, amphibian, or reptile polynucleotide or peptide.

[0408] The methods and kits disclosed herein may include the use of one or more control agents. Control agents may include control polynucleotides, inactive enzymes and/or their non-specific competitors. Alternatively, control agents include enhanced/bright hybridization agents, brightness probes, nucleic acid arrays, spike-in controls, PCR amplification controls. PCR amplification controls may be positive controls. In other cases, PCR amplification controls are negative controls. Template nucleic acid controls may be at known concentrations. Control agents may contain one or more labels.

[0409] Calibration (spike-in) controls can be matrices added to the reaction or sample. For example, a calibration template may be added to the amplification reaction. The calibration template may be added to the amplification reaction at any time after the first amplification cycle. In some embodiments, a calibration template is added to the amplification reaction after run #2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 , 45, or 50. The calibration template may be added to the amplification reaction at any time before the last amplification cycle. The calibration matrix may contain one or more nucleotides or base pairs of nucleic acids. The calibration matrix may contain DNA, RNA, or any combination thereof. The calibration matrix may contain one or more labels.

[0410] Disclosed herein are molecules, materials, compositions, and components that can be used in methods, can be used in combination with methods, can be used to prepare methods, or are products of the methods and compositions disclosed herein. It is understood that if, in the disclosure of a combination, subset, interaction, group, etc. of these materials, specific references to various individual and collective combinations and permutations of these molecules and compounds cannot be explicitly disclosed, then each of them is specifically considered and described. in this document. For example, if a nucleotide or nucleic acid is disclosed and discussed and a number of modifications are discussed that may be made to a number of molecules, including a nucleotide or nucleic acid, then each combination and permutation of the nucleotide or nucleic acid and their possible modifications are specifically considered and contemplated, unless otherwise indicated. This concept applies to all aspects of this application, including, but not limited to, steps in the methods of making and using the disclosed methods and compositions. Thus, if there are a plurality of additional steps that may be performed, it is understood that each of these additional steps may be performed in combination with any particular embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered open.

[0411] Although some of the embodiments described herein have been shown and described herein, such embodiments are presented by way of example only. Numerous variations, changes and substitutions will now be made by those skilled in the art without departing from the disclosure provided here. It should be understood that various alternatives to the embodiments described herein may be used in the implementation of the methods described herein.

[0412] Unless otherwise indicated, all technical and scientific terms used herein have the meaning/sense that is commonly understood by those skilled in the art to which this disclosure pertains. The following references contain embodiments of methods and compositions that may be applicable in this case: The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 19102); Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Mol. Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Mol. Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0413] Standard procedures of the present disclosure are described, for example, in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1989); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl (eds.), Academic Press Inc., San Diego, USA (1987)). Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed. , John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S Bonifacino et al ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), and Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998).

VII. Definitions

[0414] The terminology used here is intended only to describe specific cases and is not limited to them. Unless otherwise indicated, all terms, designations and other technical and scientific terms or terminology used in this document have the same meaning as is commonly understood by those skilled in the art to which this application pertains. In some instances, terms with conventional meanings are defined here for clarity and/or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a significant difference from what is commonly understood by those skilled in the art.

[0415] As used herein, to the extent that the terms "including", "includes", "having", "has", "with", or variations thereof are used in the detailed description and/or claims, these terms are intended to be summarized in a manner analogous to the term "comprising".

[0416] The term "about" or "approximately" can mean "within an acceptable error range" for a particular value as determined by a person skilled in the art, and depends in part on how the value is measured or determined, i.e. the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially in relation to biological systems or processes, the term may mean within the order of 5 times, and more preferably 2 times, the meaning. If specific values are described in the application and claims, and unless otherwise indicated, then the term "about" should be construed to mean "within the allowable error range for a particular value."

[0417] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues not limited by a minimum length. Polypeptides, including provided antibodies and antibody chains, and other peptides, such as linkers and binding peptides, may contain amino acid residues, including natural and/or non-natural amino acid residues. The terms also include modifications to the polypeptide after expression, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications to the native or natural sequence if the protein continues to exhibit the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as mutations in hosts that produce proteins, or errors due to PCR amplification.

[0418] Polymerase chain reaction (PCR) means an in vitro amplification reaction of polynucleotide sequences by simultaneous primer extension of the complementary strands of a double-stranded polynucleotide. PCR reactions produce copies of the template polynucleotide flanked by primer binding sites. The result of amplification with two primers is an exponential increase in the number of copies of the polynucleotide template of both strands with each cycle, because with each cycle both strands are replicated. The polynucleotide duplex has ends corresponding to the ends of the primers used. The PCR may include one or more repetitions of denaturing the template polynucleotide, annealing the primers to the primer binding site, and extending the primers with DNA or RNA polymerase in the presence of nucleotides. The specific temperatures, the duration of each cycle, and the rate of transition between cycles depend on many factors well known to those skilled in the art. (McPherson et al., IRL Press, Oxford (1991 and 1995)). For example, in conventional PCR using Taq DNA polymerase, the double-stranded template polynucleotide can be denatured at >90°C, primers can be annealed at temperatures in the range of 50-75°C, and primers can be extended at temperatures in the range of 72-78 ° C. In some embodiments, the PCR includes reverse transcription PCR (RT-PCR), real-time PCR, nested PCR, quantitative PCR, multiplex PCR, or the like. In some embodiments, the PCR does not include RT-PCR. (U.S. Patent Nos. 5,168,038, 5,210,015, 6,174,670, 6,569,627 and 5,925,517; Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002)). RT-PCR involves a PCR reaction preceded by a reverse transcription reaction and the resulting cDNA is amplified. Nested PCR includes a two-step PCR in which an amplicon of the first PCR reaction using a first set of primers becomes a template for a second PCR reaction using a second set of primers, at least one of which binds to the internal location of the first PCR reaction amplicon. Multiplex PCR includes a PCR reaction in which a plurality of polynucleotide sequences are simultaneously subjected to PCR in the same reaction mixture. PCR reaction volumes can range from 0.2 pl to 1000 µl. Quantitative PCR includes a PCR reaction designed to measure the absolute or relative amount, representation, or concentration of one or more sequences in a sample. Quantitative measurements may include comparing one or more control sequences or standards with a polynucleotide sequence of interest. (Freeman et al., Biotechniques, 26: 112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17: 9437-9447 (1989); Zimmerman et al., Biotechniques, 21: 268-279 ( 1996); Diviacco et al., Gene, 122: 3013-3020 (1992); Becker-Andre et al., Nucleic Acids Research, 17: 9437-9446 (1989)).

[0419] The terms "nucleotide", "nucleoside", "nucleotide residue" and "nucleoside residue" in the context of the present description can mean a deoxyribonucleotide or ribonucleotide residue or other analog of a nucleoside analog that can serve as a component of a primer suitable for use in the reaction amplification (for example, PCR reaction). Such nucleosides and their derivatives may be used as building blocks of the primers described herein unless otherwise indicated. Nothing in this application shall preclude the use of nucleoside derivatives or bases that have been chemically modified to increase their stability or usefulness in the amplification reaction, provided that the chemical modification does not prevent them from being recognized by the polymerase as deoxyguanine, deoxycytosine, deoxythymidine, or deoxyadenine, depending on situations. In some embodiments, the implementation of the nucleotide analogs can stabilize the formation of the hybrid. In some embodiments, nucleotide analogs can destabilize fusion formation. In some embodiments, nucleotide analogs can enhance the specificity of hybridization. In some embodiments, the implementation of nucleotide analogs can reduce the specificity of hybridization.

[0420] "Nucleic acid", or the grammatical equivalents of the term, refers to either one nucleotide or at least two nucleotides covalently linked to each other.

[0421] "Polynucleotide" or the grammatical equivalents of the term refers to at least two nucleotides covalently linked to each other. "Polynucleotide" includes a molecule containing two or more nucleotides. The polynucleotide contains a polymeric form of nucleotides of any length, or ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNA) that contain purine and pyrimidine bases, or other natural, non-natural, chemically or biochemically modified bases or derivatives of nucleotide bases. The backbone of a polynucleotide may contain sugars and phosphate groups, or modified or substituted sugar or phosphate groups. The polynucleotide may contain modified nucleotides such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. "Polynucleotide" may include other molecules, such as other hybridized polynucleotides. Polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or both sequences. Non-limiting examples of polynucleotides include gene, gene fragment, exon, intron, intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (siRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA sequences, isolated RNA sequences, nucleic acid probes and primers. Polynucleotides can be isolated from natural sources, recombinant or artificially synthesized.

[0422] Polynucleotides may include non-standard nucleotides such as nucleotide analogs or modified nucleotides. In some embodiments, the implementation of non-standard nucleotides can stabilize the formation of hybrids. In some embodiments, the implementation of non-standard nucleotides can destabilize the formation of hybrids. In some embodiments, the implementation of non-standard nucleotides can enhance the specificity of hybridization. In some embodiments, the implementation of non-standard nucleotides can reduce the specificity of hybridization. Examples of non-standard nucleotide modifications include 2'O-Me, 2'O-allyl, 2'O-propargyl, 2'O-alkyl, 2'fluoro, 2'arabino, 2'xylo, 2'fluoroarabino, phosphorothioate, phosphorodithioate, phosphoroamidates , 2'-amino, 5-alkyl-substituted pyrimidine, 3'-deoxyguanosine, 5-halo-pyrimidine, alkyl-substituted purine, halogen-substituted purine, bicyclic nucleotides, 2'MOE, PNA molecules, LNA molecules, LNA-like molecules, diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine , inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosin, 5'5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentene iladenine, uracil-5-hydroxyacetic acid (v), vivutoxosin, pseudouracil, keosin, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5 -hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2)-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and derivatives thereof.

[0423] "Subject", "individual", "host" or "patient" refers to a living organism, such as a mammal. Examples of subjects and hosts include, but are not limited to, horses, cows, camels, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice (e.g., mice expressing human genes or bearing human cells or tissues) , gerbils, non-human primates (e.g. macaques), humans and further apes, and non-mammals, including, for example, non-mammalian vertebrates such as birds (e.g. chickens or ducks), fish (e.g. sharks), or frogs (eg Xenopus), and invertebrates, as well as their transgenic species. In certain aspects, a subject refers to a single organism (eg, a human). In certain aspects, a group of individuals constituting a small cohort having either a common immune factor to study and/or disease and/or a cohort of individuals without disease (eg, negative/normal control) is provided. The sampled subject may have a disease and/or disorder (eg, one or more allergies, infections, cancer, or an autoimmune disorder, etc.) and may be compared to a negative control subject not affected by the disease.

[0424] "Kit" refers to a system for delivering materials or reagents for implementing the method disclosed in this document. In some embodiments, kits include systems that allow storage, transport, or delivery of reaction reagents (e.g., probes, enzymes, etc. in appropriate containers) and/or ancillary materials (e.g., buffers, written instructions for performing the assay) . etc.) from one place to another. For example, kits include one or more shells (eg, boxes) containing the appropriate reactive reagents and/or auxiliary materials. Such content may be delivered to the intended destination all together or separately. For example, the first container may contain an enzyme for use in the assay, while the second container contains a plurality of primers.

[0425] "Polypeptide" in some aspects refers to a molecule containing at least two amino acids. In some embodiments, the implementation of the polypeptide consists of a single peptide. In some embodiments, the implementation of the polypeptide contains two or more peptides. For example, a polypeptide may contain at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 peptides or amino acids. Examples of polypeptides include, but are not limited to: amino acid chains, proteins, peptides, hormones, polypeptide saccharides, lipids, glycolipids, phospholipids, antibodies, enzymes, kinases, receptors, transcription factors, and ligands.

[0426] "Sample" refers in some aspects to a biological, environmental, medical sample obtained from a subject or patient, or a sample containing a polynucleotide, such as a target polynucleotide.

[0427] "Pharmaceutical acceptable" refers to molecular substances and compositions that are physiologically tolerable and do not generally cause an allergic or similar adverse reaction, such as stomach upset, dizziness, and the like, when administered to a human.

[0428] "Prophylaxis" refers to prevention, preventing the onset of symptoms, preventing the progression of a disease or disorder associated with excessive protein levels or dependent on protein activity.

[0429] "Inhibition" and "treatment" are used interchangeably and refer to, for example, stabilizing/halting the progression of symptoms, prolonging life, partially or completely alleviating symptoms, and partially or completely eliminating a condition, disease, or disorder associated with excess protein or correlated with protein activity. For example, cancer treatment includes, but is not limited to, stasis, partial or complete elimination of a cancerous growth or tumor. Treatment or partial elimination includes, for example, a fold reduction in the growth or size and / or volume of the tumor, for example, about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 20 times . multiple, approximately 50-fold, or any multiple reduction. Similarly, treatment or partial elimination may include a percentage reduction in tumor growth or size and/or volume of approximately 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, 90%, 95% or any percentage reduction within these limits.

[0430] All publications, including patent documents, scientific articles and databases mentioned in this application, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were separately incorporated by reference. If the definition given here conflicts with or is otherwise inconsistent with the definition set forth in patents, applications, published applications and other publications that are incorporated here by reference, the definition set forth here takes precedence over the definition incorporated by reference.

[0431] Several aspects are described below with reference to exemplary applications for illustrative purposes. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the features described herein. However, one of ordinary skill in the relevant art will readily appreciate that the features described herein may be practiced with omission of one or more specific details, or by other means. The features described herein are not limited to the illustrated order of actions or events, as some actions may occur in a different order and/or simultaneously with other actions or events. In addition, not all illustrated actions or events are required to implement the methodology in accordance with the features described in this document.

[0432] The section heading used here is for organizational purposes only and should not be construed as limiting the subject matter being described.

VIII. Exemplary Embodiments

[0433] Among the provided embodiments are:

1. A method for obtaining a polynucleotide library, which includes adding a second adapter to each of a plurality of barcoded single stranded polynucleotides located at or near the terminal end opposite the first adapter attached to each of the barcoded single stranded polynucleotides, wherein the plurality of barcoded single stranded polynucleotides contain:

(i) one or more single-stranded target polynucleotides containing an amplicon of one or more target polynucleotides or complementary to those present in a cell from a population of cells; and

(ii) a collection of single-stranded polynucleotides, each containing an amplicon of the polynucleotide, or complementary to that, in the cell; wherein

each of the plurality of barcoded single stranded polynucleotides contains a carrier barcode which is the same for all polynucleotides of points (i) and (ii) originating from the same cell belonging to the cell population.

2. The method of Embodiment 1, wherein the plurality of single-stranded barcoded polynucleotides comprises the polynucleotides of items (i) and (ii) of a plurality of cells in a population of cells.

3. The method of Embodiment 1 or Embodiment 2, wherein each of the plurality of barcoded single stranded polynucleotides further comprises a molecular barcode that is unique to each single stranded polynucleotide.

4. The method of any one of embodiments 1-3, wherein the collection of single stranded polynucleotides from each cell belonging to the cell population collectively comprises complementary DNA (cDNA) strands of a transcriptome or partial transcriptome.

5. The method of any one of embodiments 1-4, wherein the transcriptome or partial transcriptome, in the aggregate, comprises at least 60%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97 %, 98%, 99% or 100% of the transcripts present in the cell genome.

6. The method of any one of embodiments 1-5, wherein each of the barcoded single stranded polynucleotides is greater than or about 50 base pairs (bp), greater than 100 base pairs (bp) or more than 200 base pairs (n.p.).

7. The method of any one of embodiments 1-6, wherein each of the barcoded single-stranded polynucleotides has a size ranging from or about 50 base pairs (bp) to 1500 bp, from 50 bp. P. up to 1250 b.p., from 50 b.p. up to 1000 b.p. 50 to 750 bp, 50 to 500 bp, 100 to 1500 bp, 100 to 1250 bp, 100 to 1000 bp, 100 to 750 bp .p., from 100 to 500 b.p., from 200 b.p. up to 1500 b.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p. or from 250 n.p. up to 500 b.p.

8. The method of any one of embodiments 1-7 wherein the addition of the second adapter is carried out in a homogeneous mixture containing a plurality of barcoded single strand polynucleotides.

9. The method of any one of embodiments 1-8, wherein the first adapter contains a media barcode.

10. A method for obtaining a polynucleotide library, including:

(a) lysing a cell in each of a plurality of media, where each of these media contains a cell from a sample containing a population of cells;

(b) producing in each carrier a plurality of complementary polynucleotides, said production of said plurality of complementary polynucleotides comprising (i) producing one or more target polynucleotides that are complementary to one or more target polynucleotides present in the cell using one or more target-specific primers; and (ii) obtaining a collection of polynucleotides, each of which is complementary to a polynucleotide in the cell, using random oligoprimers.

11. The method of embodiment 10, wherein each of said carriers further comprises a plurality of oligonucleotides carrying a molecular barcode, a single or pool of barcoded oligonucleotides, and optionally a first adapter, the method further comprising:

(c) attaching to a plurality, optionally each of the plurality of complementary polynucleotides, one of the plurality of barcoded molecular oligonucleotides, thereby generating a plurality of barcoded molecular polynucleotides, each containing a molecular barcode, where optionally a molecular barcode the code of each of the molecular barcoded polynucleotides is different from the molecular barcodes contained in other molecular barcoded polynucleotides from the plurality and/or is a unique molecular barcode;

(d) attaching one of the single or pool of carrier barcoded oligonucleotides and the first adapter or amplified product thereof to a plurality, or optionally each of the barcoded polynucleotides, resulting in a plurality of double-barcoded polynucleotides -code, where each of the double-barcoded polynucleotides of the same carrier contains the same carrier barcode.

12. The method of carrying out the invention according to claim 11, further comprising (e) obtaining a single-stranded amplicon from a plurality, optionally each of the plurality, of double-barcoded polynucleotides.

13. The method of carrying out the invention according to 11, further comprising (f) adding a second adapter to each of the single stranded amplicons, wherein the first adapter and the second adapter are located at or approximately opposite ends of each of the single stranded double tri-code polynucleotides.

14. A method for obtaining a polynucleotide library, including:

(a) lysing a cell in each of a plurality of carriers, wherein each of said carriers comprises a cell from a sample containing a population of cells, a plurality of molecular barcode oligonucleotides and a single or pool of carrier barcode oligonucleotides, and optionally a first adapter;

(b) producing in each carrier a plurality of complementary polynucleotides, said producing said plurality comprising (i) producing one or more target polynucleotides complementary to one or more target polynucleotides present in the cell; and (ii) obtaining a collection of polynucleotides, each of which is individually complementary to a polynucleotide in the cell;

(c) attaching to each complementary polynucleotide one of a plurality of oligonucleotides with a molecular barcode, thereby generating a plurality of polynucleotides with a barcode, each of which contains a unique molecular barcode;

(d) attaching one of a single or pool of carrier-barcoded oligonucleotides, or amplified products thereof, to each of the barcoded polynucleotides, resulting in a plurality of double-barcoded polynucleotides, wherein each of the double-barcoded polynucleotides located in the same media, contains the same media barcode;

(e) obtaining a single-stranded amplicon of each of the plurality of polynucleotides with two barcodes; and

(f) adding a second adapter to each of the single-stranded amplicons, which means attaching the second adapter to the double-barcoded single-stranded polynucleotide, with the first adapter and the second located at or approximately opposite ends of each of the double-barcoded single-stranded polynucleotides.

15. The method of any one of embodiments 11-14, wherein the first adapter comprises an oligonucleotide with a carrier barcode.

16. The method of any one of embodiments 10-15, wherein the polynucleotide pool of each cell of the cell population collectively comprises sequences that are complementary to transcriptome or partial transcriptome transcripts of the cell.

17. The method of any one of embodiments 16, wherein the transcriptome or partial transcriptome comprises at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% of transcripts present in the cell genome.

18. The method of any one of embodiments 1-17, wherein the one or more target polynucleotides and/or polynucleotides in the cell is DNA.

19. The method of any one of embodiments 1-18, wherein the one or more target polynucleotides and/or polynucleotides in the cell is RNA.

20. The method according to claim 19, wherein the RNA is mRNA.

21. The method of any one of embodiments 10-20, wherein each of the single or more complementary polynucleotides (b) is cDNA.

22. The method of any one of embodiments 1-21, wherein each or one or more of the barcoded single stranded polynucleotides is a cDNA strand.

23. The method of any one of embodiments 1-22, wherein the first adapter and/or the second adapter comprise at least one universal primer.

24. The method of any one of embodiments 1-23, wherein:

the first adapter and the second adapter are different; and/or

the first adapter contains the first universal priming site, and the second adapter contains the second universal priming site, and where, optionally, the first universal priming site and the second universal priming site are different.

25. The method of embodiment 24, wherein the first universal priming site and/or the second universal priming site is or contains a P7 (C7) priming site or a continuous portion thereof, or a P5 (C5) priming site or a continuous portion thereof, and where, optionally, the continuous portion is sufficient for annealing with a complementary sequence.

26. The method of embodiment 24 or embodiment 25, wherein the first universal priming site is or contains a P7 (C7) priming site or a contiguous portion thereof, and the second universal priming site is or contains a P5 (C5) priming site or a contiguous portion thereof. continuous part.

27. The method of embodiment 25 or embodiment 26, wherein the P7 (C7) priming site contains or is a contiguous portion of the sequence AGATCGGAAGAGCACACGTCTGAACTCCA (SEQ ID NO: 77).

28. The method of Embodiment 25 or Embodiment 26, wherein the P5 priming site contains or is a contiguous portion of the sequence AGATCGGAAGAGCGTCGTGTGTAGGGAAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT (SEQ ID NO: 78).

29. The method of any one of embodiments 25-28, wherein the contiguous portion is at least or at least about 15, 20, 25, or 30 nucleotides in length.

30. The method of any one of embodiments 25, 26, 28, or 29, wherein the P5 priming site is a contiguous portion as indicated in SEQ ID NO: 25 (AGATCGGAAGAGCGTCGTGT).

31. The method of any one of embodiments 1-9 and 13-30, wherein the addition of the second adapter comprises hybridizing a hinged (splint) oligonucleotide to each of the barcoded single-stranded polynucleotides in the presence of an oligonucleotide containing a second universal priming site, where hinge ( splint) the oligonucleotide comprises (i) a sequence complementary to the second universal priming site and (ii) a degenerate overhang sequence capable of randomly annealing the 3' end of the single-stranded barcode polynucleotide.

32. The method of embodiment 31 wherein, prior to hybridization, the splint oligonucleotide and the oligonucleotide containing the second universal priming site are annealed to form an adapter duplex with the splint oligonucleotide.

33. The method of embodiment 31 or 32 wherein the degenerate overhang sequence comprises the sequence (N) _3-12 where N is any nucleotide.

34. The method of any one of embodiments 31-33, wherein the degenerate overhang sequence comprises the sequence NNNNNN, where N is any nucleotide (SEQ ID NO: 24).

35. The method of any one of embodiments 31-34, wherein the splint oligonucleotide comprises the sequence ACACGACGCTCTTCCGATCTNNNNNN, where N is any amino acid (SEQ ID NO: 26).

36. The method of any one of embodiments 31-35, wherein the oligonucleotide containing the second universal priming site contains the sequence AGATCGGAAGAGCGTCGTGT (SEQ ID NO: 25).

37. The method of any of embodiments 1-9 and 11-36, wherein said carrier-barcoded oligonucleotide comprises at least or at least about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 nucleotides.

38. The method of any one of embodiments 1-9 and 11-37, wherein said carrier-barcoded oligonucleotide contains from or about 10 to 30 nucleotides.

39. The method of any one of embodiments 1-38, wherein said carrier barcoded oligonucleotide contains a degenerate sequence.

40. The method of any one of embodiments 1-9 and 11-39, wherein said carrier barcoded oligonucleotide comprises the sequence (N) _14-17 , where N is any nucleotide, and where, optionally, at least one or two N's in sequence are W, where W is adenine or thymine.

41. The method of any one of embodiments 1-9 and 11-40, wherein said carrier barcode oligonucleotide comprises the sequence NNNNWNNNNWNNNN (SEQ ID NO: 80), WNNNNWNNNNWNNNN (SEQ ID NO: 81), NWNNNWNNNNWNNNN: SEQ ID, or NNWNNNNWNNNNWNNNN (SEQ ID NO: 83), and where N is any nucleotide and W is adenine or thymine.

42. The method of any one of embodiments 11-41, wherein each carrier comprises a pool of first adapters, wherein each carrier-barcoded oligonucleotide from the pool of first adapters contains at least one displaced base or added base compared to at least one from other oligonucleotides with a carrier barcode in the pool.

43. The method of embodiment 42 wherein the carrier barcoded oligonucleotides from the pool of first adapters comprise the sequences NNNNWNNNNWNNNN (SEQ ID NO: 80), WNNNNWNNNNWNNNN (SEQ ID NO: 81), NWNNNWNNNNWNNNN (SEQ ID NO: 82), and NNWNNNNWNNNNWNNNN (SEQ ID NO: 83) where N is any nucleotide and W is adenine or thymine.

44. The method of any one of embodiments 11-43, wherein in step (d) the method further comprises amplifying a single or pool of carrier-barcoded oligonucleotides, a pool of first adapters comprising a single or pool of carrier-barcoded oligonucleotides, and wherein amplifying carried out before or simultaneously with the attachment of the oligonucleotide with a carrier barcode.

45. The method of any one of embodiments 11-44, wherein the attachment of the carrier barcode oligonucleotide comprises hybridizing a region of the carrier barcode oligonucleotide with a region of each of the complementary polynucleotides or with a region of each of the molecular barcode polynucleotides containing a molecular bar -code.

46. The method of embodiment 45 wherein the region comprises a 3' labeled/labeled polynucleotide complementary to the 5' molecular barcode region of the molecular barcode polynucleotides.

47. The method of any one of embodiments 10-46, wherein in step (b):

one or more target polynucleotides are obtained by reverse transcription of the target polynucleotide(s) in the presence of reverse transcriptase and one or more target-specific primers complementary to the target sequence of the target polynucleotide(s); and/or

a collection of polynucleotides is produced by reverse transcription of the polynucleotides in the cell in the presence of a reverse transcriptase and one or more transcriptome primers complementary to the polynucleotide in the cell.

48. The method of any one of embodiments 1-47, wherein the one or more target polynucleotides comprise an immune molecule polynucleotide or chain thereof.

49. The method of any one of embodiments 1-48, wherein the one or more target polynucleotides comprise at least two target polynucleotides each containing an immune system molecule chain polynucleotide.

50. The method of any one of embodiments 1-49, wherein the one or more target polynucleotides comprise a TCR polynucleotide or chain thereof.

51. The method of any one of embodiments 1-50, wherein one or more of the target polynucleotides comprises a T cell receptor alpha (TCRα) polynucleotide and the second is a T cell receptor (TCRβ) polynucleotide.

52. The method of any one of embodiments 1-50, wherein one or more of the target polynucleotides comprises a T cell receptor gamma (TCRγ) polynucleotide and the second is a T cell receptor delta (TCRdelta) polynucleotide.

53. The method of any one of embodiments 1-49, wherein the one or more target polynucleotides comprise an antibody polynucleotide or chains thereof.

54. The method of any one of embodiments 1-49 and 53, wherein the one or more target polynucleotides comprise one immunoglobulin heavy chain (IgH) polynucleotide and the second polynucleotide is an immunoglobulin light chain (IgL) polynucleotide.

55. The method of any one of embodiments 47-54, wherein one or more target-specific primers and/or one or more transcriptome primers comprise a poly(T) sequence.

56. The method of any one of embodiments 47-54, wherein the one or more transcriptome primers comprise a mixture of random hexamer oligonucleotide primers.

57. The method of any one of embodiments 47-56, wherein the one or more target-specific primers comprise one or more primers that are complementary to the sequence(s) of the target sequence(s) of the target polynucleotide.

58. The method of embodiment 57, wherein the one or more target-specific primers comprise at least a first primer and a second primer.

59. The method of embodiment 57 or embodiment 58, wherein the one or more target-specific primers include primers for the target sequence(s) of a plurality of immune molecules or chains thereof.

60. The method of embodiment 59 wherein the immune molecule is a T cell receptor or an antibody.

61. The method of any one of embodiments 58-60, wherein at least the first primer is complementary to the target polynucleotide sequence of the first chain of the immune molecule, and the second primer is complementary to the target sequence of the polynucleotide of the second chain of the immune molecule.

62. The method of any one of embodiments 58-61, wherein the first and second primers are complementary to the target sequences of different TCR strands of TCR polynucleotides.

63. The method of any one of embodiments 58-62, wherein:

the first primer is complementary to the target sequence of the TCRalpha polynucleotide sequence and the second primer is complementary to the target sequence of the TCRbeta polynucleotide sequence; or

the first primer is complementary to the target sequence of the TCRgamma polynucleotide sequence, and the second primer is complementary to the target sequence of the TCRdelta polynucleotide sequence.

64. The method of embodiment 62 or embodiment 63, wherein the target polynucleotide sequence of the TCR chain is a constant region sequence.

65. The method of any one of embodiments 58-64, wherein:

the first primer is complementary to the target constant region of the TCR-alpha polynucleotide sequence, and the second primer is complementary to the target constant region of the TCR-beta polynucleotide sequence; or

the first primer is complementary to the target constant region of the TCRgamma polynucleotide sequence, and the second primer is complementary to the target constant region of the TCRdelta polynucleotide sequence.

66. The method of any one of embodiments 58-61, wherein at least the first and second primers are complementary to target sequences of polynucleotide sequences of different antibody chains.

67. The method of any one of embodiments 58-61 and 66 wherein the first primer is complementary to a target sequence present in an immunoglobulin heavy chain (IgH) polynucleotide sequence and the second primer is complementary to a target sequence present in a light chain polynucleotide sequence. immunoglobulin (IgL).

68. The method of embodiment 66 or embodiment 67, wherein the target sequence of the polynucleotide sequences of the antibody chains is a constant region sequence.

69. The method of any one of embodiments 58-61 and 66-68, wherein the first primer is complementary to the heavy chain constant region (CH) polynucleotide target sequence and the second primer is complementary to the light chain constant region (CL) target sequence.

70. The method of embodiment 68 or embodiment 69, wherein:

the target sequence of the CH polynucleotide is from IgM, IgD, IgA, IgE, or IgG, or a combination thereof; and/or

the target sequence of the CL polynucleotide sequence is from Igkappa, Iglambda, or combinations thereof.

71. The method of any one of embodiments 1-70, wherein the one or more target polynucleotides is a full-length coding sequence.

72. The method of any one of embodiments 10-71, wherein one or more target polynucleotides and a collection of polynucleotides are generated in a vehicle in the same reaction volume.

73. The method of any one of embodiments 10-72, wherein in step (b) the production of a plurality of complementary polynucleotides comprises the use of a non-template terminal transferase, and wherein three or more non-template nucleotides, ribonucleotides, or analogues thereof are added to the 3' end of each produced complementary polynucleotide.

74. The method of embodiment 73 wherein the non-template terminal transferase is a reverse transcriptase or polymerase.

75. The method of Embodiment 73 or Embodiment 74 wherein the non-template terminal transferase is a reverse transcriptase and wherein the reverse transcriptase is selected from the list of Superscript II reverse transcriptase, Maxima reverse transcriptase, Protoscript II reverse transcriptase, Maloney murine leukemia virus reverse transcriptase. transcriptase (MMLV-RT), HighScriber reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, any reverse transcriptase having terminal deoxynucleotidyltransferase activity, and combinations thereof.

76. The method of any one of embodiments 11-75, wherein in step (c) the addition comprises hybridizing a region of one of the oligonucleotides carrying a molecular barcode with three or more non-template nucleotides of each of the complementary polynucleotides.

77. The method of any one of embodiments 73-75, wherein the plurality of molecular barcode oligonucleotides are provided as a plurality of template-switched oligonucleotides, each containing a 3' portion complementary to three or more non-template nucleotides.

78. The method of embodiment 77, wherein the template switch oligonucleotide also contains a 5' end region complementary to the 3' labeled oligonucleotide of the first adapter containing the carrier barcode.

79. The method of any one of embodiments 11-78, wherein:

reverse transcriptase has template switching activity;

at least some strands of the plurality of complementary polynucleotides obtained contain a 3' overhang containing three or more non-template nucleotides;

the plurality of molecular barcode oligonucleotides are presented as a plurality of template-switched oligonucleotides, each containing (1) a 5' end region complementary to the 3' region of the labeling oligonucleotide of the first adapter containing the carrier barcode, (2) a molecular barcode code and (3) a 3'-part complementary to three or more non-template nucleotides of the 3'-overhang; and where

the template switch oligonucleotide serves as a template for reverse transcriptase such that a molecular barcode is included in each complementary polynucleotide.

80. The method of any one of embodiments 77-79, wherein the 3' region complementary to three or more non-template nucleotides comprises a nucleotide, ribonucleotide, or analog thereof.

81. The method of any one of embodiments 73-80, wherein the three or more non-template nucleotides comprise three or more C nucleotides and the 3' portion complementary to the three or more non-template nucleotides contains one or more G nucleotides or ribonucleotides or analogs thereof. .

82. The method of any one of embodiments 73-77, wherein the template switch oligonucleotide further comprises a 3'-modified nucleotide that blocks elongation by reverse transcriptase or DNA polymerase.

83. The method of embodiment 82 wherein the modification is a deoxy, phosphate, amino, or alkyl modification of the 3'-nucleotide.

84. The method of any one of embodiments 11-83, wherein step (d) further comprises extending each of the plurality of complementary polynucleotides bearing the molecular barcode after addition.

85. The method of any one of embodiments 1-84, wherein the carrier is a well contents, an emulsion, or a drop.

86. The method of any one of embodiments 12-85, comprising, prior to step (e), combining the contents of two or more of the plurality of carriers, and thereby creating a homogeneous mixture containing two or more of the plurality of double stranded single stranded polynucleotides. code.

87. The method of embodiment 86, wherein combining the contents of a plurality of carriers comprises disrupting two or more of the plurality of carriers and combining double-barcoded single-stranded polynucleotides from the two or more selected carriers.

88. The method of embodiment 86 or embodiment 87, comprising, prior to step (e), selecting or purifying double-barcoded single-stranded polynucleotides having a size greater than or about greater than 50 base pairs, greater than 100 base pairs, or greater than 200 base pairs.

89. The method of any one of embodiments 86-88 comprising, prior to step (e), selecting or purifying double-barcoded single-stranded polynucleotides having a size of from or about 50 base pairs (bp) to 1500 bp. , from 50 n.p. up to 1250 bp, from 50 to 1000 bp, from 50 to 750 bp, from 50 to 500 bp, from 100 to 1500 bp, from 100 to 1250 bp ., from 100 to 1000 b.p., from 100 to 750 b.p., from 100 b.p. up to 500 b.p. from 200 n.p. up to 1500 b.p., from 200 b.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p. or from 250 n.p. up to 500 b.p.

90. The method of any one of embodiments 1-89, wherein the double-barcoded single-stranded polynucleotides are in order (5' to 3'): first adapter, carrier barcode, molecular barcode, and second adapter.

91. The method of any one of embodiments 1-90, wherein the first adapter is located at or about 5' region of the double-barcoded single stranded polynucleotide.

92. The method of any one of embodiments 1-91, wherein the second adapter is located at or about the 3' region of the double-barcoded single stranded polynucleotide.

93. The method of any one of embodiments 10-92, wherein one or more of steps (a)-(f) is carried out in solution and in the presence/absence of a solid support, optionally a granule.

94. The method of any one of embodiments 11-93, wherein at least steps (c) and (d) are carried out in solution and in the presence/absence of a solid support, optionally a granule.

95. The method of any one of embodiments 10-94, wherein each of steps (a)-(e) is carried out in solution and in the presence/absence of a solid support, optionally a granule.

96. The method of any one of embodiments 1-95, wherein the cell population comprises at least or about at least 1×10 ³ , 5×10 ³ , 1×10 ⁴ , 5×10 ⁴ , 1×10 ⁵ , 5 ×10 ⁵ , 1×10 ⁶ , or 5×10 ⁶ cells.

97. The method of any one of embodiments 1-96, wherein the cell population is derived from a biological sample from a subject.

98. The method of embodiment 97 wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product .

99. The method of any one of embodiments 1-98, wherein the cell population comprises immune cells.

100. The method of any one of embodiments 1-99, wherein the immune cells include lymphocytes or antigen presenting cells.

101. The method of any one of embodiments 1-100, wherein the immune cell is a lymphocyte or subtype thereof, a B cell or subtype, a T cell or subtype thereof, or a combination thereof.

102. The method of embodiment 101 wherein the immune cell is a T cell that is a CD4+ and/or CD8+ T cell.

103. The method of any one of embodiments 1-102, wherein the cell population is enriched in or includes central memory T cells, effector memory T cells, naïve T cells, stem central memory T cells, effector T cells, and regulatory T cells. cells.

104. The method of any one of embodiments 1-101, wherein the cell population is enriched for memory B cells, naive B cells, or plasmablast B cells.

105. The method of any one of embodiments 97-104, wherein the subject is a human.

106. The method of any one of embodiments 97-105, wherein the subject has a cancer, infection, or autoimmune condition.

107. The method of embodiment 106 wherein the infection is a viral, bacterial, or fungal infection.

108. The method of any one of embodiments 1-107, further comprising amplifying a plurality of barcoded single strand polynucleotides, and thereby generating a plurality of polynucleotide templates.

109. The method of any one of embodiments 1-108, wherein the amplification is performed in the presence of a first primer set comprising a first primer complementary to the first adapter sequence and a second primer complementary to the second adapter sequence.

110. The method of embodiment 109 wherein the first and/or second primer is a universal primer.

111. The method of embodiment 110 wherein the first and/or second primer is complementary to a P7 priming site (C7) or a contiguous portion thereof or a P5 primer site (C5) or a continuous portion thereof.

112. The method of embodiment 110 or embodiment 111 wherein the first primer is complementary to the P7(C7) priming site or a contiguous portion thereof and the second primer is complementary to the P5(C5) priming site or a continuous portion thereof.

113. The method of embodiment 111 or embodiment 112, wherein:

the primer complementary to the P7(C7) priming site, or a contiguous portion thereof, has or contains the sequence CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 39); and/or

the primer complementary to the P5 priming site (C5) or a contiguous portion thereof contains the sequence ACACGACGCTCTTCCGATCT (SEQ ID NO: 27).

114. The method of any one of embodiments 109-113, wherein the first and/or second primer further comprises a sequencing adapter.

115. The method of embodiment 114, wherein:

a primer that is complementary to the P7(C7) primary site, or a contiguous portion thereof, further comprises the sequence CAAGCAGAAGACGGCATACGAGAT [NNNNNN] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 28); and/or

the primer complementary to the P5 (C5) priming site or a contiguous portion thereof contains the sequence AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC (SEQ ID NO: 76).

116. The method of embodiment 115, further comprising purifying each of the plurality of barcoded polynucleotides.

117. A polynucleotide library containing a plurality of barcoded polynucleotides obtained by the method of any of embodiments 1-116.

118. A polynucleotide library containing a plurality of barcoded polynucleotides, where the plurality of barcoded polynucleotides comprises (i) one or more target polynucleotides containing an amplicon of one or more target polynucleotides present in a cell belonging to a population of cells; and (ii) a collection of polynucleotides, each containing an amplicon of the polynucleotide in the cell, each barcoded polynucleotide containing:

a first adapter containing a first universal priming site complementary to the first universal primer;

a carrier barcode that is the same for all barcoded polynucleotides of items (i) and (ii) originating from the same cell belonging to the cell population; and

a second adapter sequence comprising a second universal priming site complementary to the second universal primer.

119. The polynucleotide library of embodiment 118, wherein each of the plurality of barcoded polynucleotide templates contains a molecular barcode that is unique to each polynucleotide template.

120. The polynucleotide library of embodiment 118 or embodiment 119, wherein the array of barcoded polynucleotide templates of each cell of the cell population collectively comprises complementary DNA (cDNA) strands of a transcriptome or partial transcriptome.

121. The polynucleotide library of any one of embodiments 118-120, wherein the transcriptome or partial transcriptome together comprise at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the transcripts present in the cell's genome.

122. The polynucleotide library of any one of embodiments 118-121, wherein each of the barcoded polynucleotide templates has a size that is greater than or about greater than 50 base pairs, greater than 100 base pairs, or greater than 200 base pairs.

123. The polynucleotide library of any one of embodiments 118-122, wherein each of the barcoded single stranded polynucleotides is from or about 50 base pairs (bp) to 1500 bp, from 50 bp. up to 1250 b.p., from 50 b.p. up to 1000 b.p., from 50 b.p. up to 750 b.p., from 50 b.p. up to 500 b.p., from 100 b.p. up to 1500 b.p., from 100 b.p. up to 1250 b.p., from 100 b.p. up to 1000 b.p., from 100 b.p. up to 750 b.p., from 100 b.p. up to 500 b.p., from 200 b.p. up to 1500 b.p., from 200 b.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p. or from 250 n.p. up to 500 b.p. steam.

124. The polynucleotide library of any one of embodiments 118-123, wherein the first adapter contains a carrier barcode.

125. The polynucleotide library of any one of embodiments 118-124, wherein the first adapter and the second adapter are different.

126. The polynucleotide library of any one of embodiments 118-125, wherein the first universal priming site and/or the second universal priming site is or contains a P7(C7) priming site, or a contiguous portion thereof, or a P5(C5) priming site, or a a continuous portion, and where, optionally, a continuous portion thereof is sufficient for annealing with a complementary sequence.

127. The polynucleotide library of any one of embodiments 118-126, wherein the first universal priming site is or contains a P7(C7) priming site or a contiguous portion thereof, and the second universal priming site is or contains a P5(C5) priming site or a continuous portion thereof. Part.

128. A polynucleotide library according to variant 126 or variant 127, in which the P7(C7) priming site contains the sequence AGATCGGAAGAGCACACGTCTGAACTCCA (SEQ ID NO: 77) or is a contiguous part of it.

129. A polynucleotide library according to version 126 or according to version 127, in which the P5 priming site contains the sequence AGATCGGAAGAGCGTCGTGTAGGGAAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT (SEQ ID NO: 78) or is a contiguous part of it.

130. The polynucleotide library of any one of embodiments 126-129, wherein the contiguous portion is at least or at least about 15, 20, 25, or 30 nucleotides in length.

131. The polynucleotide library of embodiment 129 or 130 wherein the P5 priming site is a contiguous portion as indicated in SEQ ID NO: 25 (AGATCGGAAGAGCGTCGTGT).

132. The polynucleotide library of any one of embodiments 118-131, wherein the carrier-barcoded oligonucleotide contains at least or about at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 nucleotides.

133. The polynucleotide library of any one of embodiments 118-132, wherein the oligonucleotide containing the carrier barcode contains from or about 10 to 30 nucleotides.

134. The polynucleotide library of any one of embodiments 118-133, wherein the one or more target polynucleotides comprise an immune molecule or chain polynucleotide.

135. The polynucleotide library of any one of embodiments 118-134, wherein the one or more target polynucleotides comprise at least two target polynucleotides, each containing an immune molecule chain polynucleotide.

136. The polynucleotide library of any one of embodiments 118-135, wherein the one or more target polynucleotides is a TCR polynucleotide or chain thereof.

137. The polynucleotide library of any one of embodiments 118-136, wherein the one or more target polynucleotides are a T cell receptor alpha (TCRα) polynucleotide and a T cell receptor (TCRβ) polynucleotide.

138. The polynucleotide library of any one of embodiments 118-136, wherein the one or more target polynucleotides are a T cell receptor gamma (TCRγ) polynucleotide and a T cell receptor delta (TCRdelta) polynucleotide.

139. The polynucleotide library of any one of embodiments 118-135, wherein the one or more target polynucleotides comprise an antibody polynucleotide or chains thereof.

140. The polynucleotide library of any of embodiments 118-135 and 139, wherein the one or more target polynucleotides comprise a first polynucleotide that is an immunoglobulin heavy chain (IgH) polynucleotide and a second polynucleotide that is an immunoglobulin light chain (IgL) polynucleotide.

141. The polynucleotide library of any one of embodiments 118-140, wherein the barcoded polynucleotides are in order (5' to 3'): first adapter, carrier barcode, molecular barcode, and second adapter.

142. The polynucleotide library of any one of embodiments 118-1141, wherein the first adapter is located at or about the 5' region of the double-barcoded single-stranded polynucleotide.

143. The polynucleotide library of any one of embodiments 118-137, wherein the second adapter is located at or about the 3' region of the double-barcoded single-stranded polynucleotide.

144. A sequencing method comprising sequencing one or more of a plurality of barcoded polynucleotides obtained according to any one of embodiments 1-116 or from a polynucleotide library according to any one of embodiments 118-141.

145. The method of embodiment 144 wherein a transcriptome derived from a plurality of barcoded polynucleotides is sequenced.

146. The method of embodiment 145 further comprising amplifying all or part of the transcriptome prior to sequencing.

147. The method of embodiment 146 wherein the amplification is performed using a first primer set comprising a first primer and a second primer specific to the first and second adapter sequences, respectively.

148. The method of embodiment 146 or 147 wherein one or more target polynucleotides from a plurality of barcoded polynucleotides are sequenced.

149. The method of embodiment 148, further comprising amplifying one or more target polynucleotides from a plurality of polynucleotide templates prior to sequencing.

150. The method of embodiment 149 wherein the full length sequence(s) of one or more target polynucleotides are amplified.

151. The method of embodiment 149 or 150 wherein the amplification is performed in the presence of a second primer set comprising one or more first primers complementary to one or more target polynucleotides and a second primer complementary to the first adapter sequence.

152. The method of embodiment 151, wherein the second primer of the second primer set is complementary to the P7(C7) priming site, or a contiguous portion thereof, or the P5(C5) primer site, or a continuous portion thereof.

153. The method of embodiment 151 or 152, wherein the second primer of the second primer set is complementary to the P7(C7) priming site or a contiguous portion thereof.

154. The method of any one of embodiments 151-153, wherein the second primer of the second primer set has or includes the sequence CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 39) or CAAGCAGAAGACGGCATACGAGAT [NNNNN] GTGACTGGAGTTCAGACGTGTGCTCTTQGCT: 28).

155. The method of any one of embodiments 151-154, wherein the one or more first primers complementary to one or more target polynucleotides are specific for a target sequence of an immune molecule or chain thereof.

156. The method of embodiment 155 wherein the immune system molecule is a T cell receptor or an antibody.

157. The method of embodiment 155 or 156 wherein the one or more first primers are specific for the target sequence of the constant region of the immune molecule.

158. The method of any one of embodiments 155-157 wherein the immune molecule is a TCR and the one or more first primers comprise AGTCTCTCAGCTGGTACACGG (SEQ ID NO: 37), ATGGCTCAAACACAGCGACCTC (SEQ ID NO: 38), or a combination thereof.

159. The method of any one of embodiments 155-158, wherein the immune molecule is an antibody and one or more first primers comprise any of SEQ ID NOs: 29-36 or a combination thereof.

160. The method of any one of embodiments 144-159, comprising identifying the cell from which one or more barcoded polynucleotides originate.

161. The method of embodiment 160 wherein identifying the cell from which the polynucleotides originate includes obtaining information about sequences having the same carrier barcode as originating from the same cell.

162. The method of any one of embodiments 144-161, wherein the target polynucleotide is an immune molecule having a first polynucleotide strand and a second polynucleotide strand, and the method comprises grouping the first polynucleotide strand and the second polynucleotide strand of the same cell based on the presence of one and the same media barcode.

163. The method of any one of embodiments 154-162, which also includes quantifying or determining the number of polynucleotides having the same molecular barcode.

164. The method of any one of embodiments 154-163, wherein the method comprises identifying transcriptome sequences and target polynucleotide sequences having the same carrier barcode, thereby providing information about the transcriptome of the cell bearing the target polynucleotide(s) .

165. A method for analyzing a transcriptome, including:

(a) sequencing a target polynucleotide from barcoded polynucleotides belonging to a plurality of barcoded polynucleotides obtained by the method of any of embodiments 1-116, or from a plurality of barcoded polynucleotides belonging to a polynucleotide library of any of embodiments 118-141 , thereby generating target polynucleotide sequence information from a plurality of cells;

(b) sequencing all or part of the transcriptome from a plurality of barcoded polynucleotides obtained by the method of any one of embodiments 1-116, or from a plurality of barcoded polynucleotides of a polynucleotide library according to any one of embodiments 118-141, and thereby generating transcriptome data from the plurality cells; and

(c) identifying sequence information from (a) and from (b) that have the same carrier barcode as originating from the same cell.

166. A method for analyzing the transcriptome of a selected individual cell, including:

(a) amplifying and sequencing a target polynucleotide from a plurality of barcoded polynucleotides obtained by the method of any of embodiments 1-116, or from a plurality of barcoded polynucleotides obtained from a polynucleotide library of any of embodiments 118-141, thereby generating information about sequences for each target polynucleotide in at least one of the plurality of cells;

(b) identifying the carrier barcode(s) associated with one of the target polynucleotides sequenced in (a), thereby identifying the selected individual cell carrying the target polynucleotide;

(c) amplifying and sequencing the transcriptome, or a portion thereof, based on the plurality of barcoded polynucleotides of the cell carrying the carrier barcode, thereby generating transcriptome data from the selected cell expressing the target polypeptide.

167. The method of embodiment 166 wherein a transcriptome or portion thereof from a selected cell is amplified or sequenced using a primer specific for the carrier barcode specified in (b) and a primer specific for the second sequence of the barcoded polynucleotide adapter.

168. A method for analyzing a transcriptome, which includes matching information about the sequence of a transcriptome or a part thereof and at least one or more of the target polynucleotides that originate from the same cell, while the sequence information is either determined based on a plurality of barcoded polynucleotides obtained by the method of any of embodiments 1-114 or based on a plurality of polynucleotide templates of a polynucleotide library of any of embodiments 118-141, or determined by the method of any of embodiments 154-164.

169. The method of embodiment 168 wherein sequences that have the same carrier barcode are grouped as being in the same cell.

170. The method of any one of embodiments 165-169, wherein the transcriptome data comprises a parameter, characteristic, trait, or phenotype associated with cell function or activity.

171. The method of embodiment 170, wherein the transcriptome data is associated with cell activation, depletion, or proliferative activity.

IX. Examples

[0434] The following examples are included for illustrative purposes only and should not be construed as limiting the scope of the invention.

Example 1. Barcoding of transcripts in emulsion for sequencing of single cell polynucleotides.

A. Cell preparation

[0435] Single cell suspensions for single cell polynucleotide sequencing were obtained from peripheral blood mononuclear cells (PBMCs). Approximately 50 mL of blood was drawn into Vacutainer CPT Cell Preparation Tubes for cell preparation with sodium heparin (BD), centrifuged for 20 minutes at 1800×g, washed twice in cell preparation buffer (1x PBS supplemented with 2% fetal bovine serum and 2 mM EDTA), twisted at 200 × g to remove platelets, and the resulting PBMCs were cryopreserved in RPMI-1640 (Life Technologies) + 20% fetal bovine serum + 10% DMSO at -80 °C until further operations. PBMCs were thawed before emulsion generation, washed twice in cell buffer: 1 × Dulbecco's phosphate-buffered saline (PBS) with centrifugation (200 × g for 10 min). The cells were then diluted in cell buffer to a cell concentration of 3.5×10 ⁶ cells/ml. The suspension was then pipetted through a 20 μm cell filter.

B. Barcoding in emulsion

[0436] An emulsion was formed containing the prepared cells and the reaction mixture. The reaction mixture was prepared as a 2-fold concentrate, which was mixed in a 1:1 volume ratio with the cell suspension during the droplet formation process.

1. Preparation of the emulsion reaction mixture

[0437] The emulsion reaction mixture containing the reagents and oligonucleotides in Table E1 below was mixed at room temperature in a fume hood in which no PCR was performed.

Table E1. Emulsion reaction mixture Reagent Tris-Cl, pH 8.0 _MgSO4 DTT dNTPs each 5'biotn oligo-dT Matrix switching, oligonucleotide VB matrix molecules/µl VB primer straight VB primer reverse Protease inhibitor (X) Enzymatic RNase Inhibitor (U/µl ) MMLV RNase H-reverse transcriptase DNA polymerase Triton X-100 (%v/v) Water Oligonucleotide sequences 5′ biotin oligo-dT anchored reverse transcription primer /5BiosG//iSpl8/TTT TTT TTT TTT TTT TTT TTT TTT TVN
(SEQ ID NO:1) Media Barcode Matrix ATCCATCCACGACTGACGGACGTATTAAANNNNWNNNNWNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACC
(SEQ ID NO:2) Template switch oligonucleotide AATACGTCCGTCAGTCGTGGATGNNTNNANNTrGrGG
(SEQ ID NO:3) Barcode media direct (primer) CATCCACGACTGACGGACGTATT
(SEQ ID NO:4) Reverse media barcode (primer) GTGACTGGAGTTCAGACGTGTGCT
(SEQ ID NO:5) /5BiosG/ = 5' modification with biotin
/iSpl8/ = 18-carbon spacer
V=A, C, or G
N=any base
rG=riboguanosine
W=A or T

2. Creation of double-barcoded libraries based on single cells

[0438] An overview of an exemplary method for generating dual barcode polynucleotide libraries is shown in FIGS. 1A and 1B. The emulsion was obtained using the prepared cells and the reaction mixture. The emulsion generation platform included three Mitos P-Pumps (Dolomite Microfluidics) driven by a single air compressor, each pump equipped with a Mitos Flow Rate sensor to provide computer controlled flow of two aqueous phases and one continuous fluoro oil phase Dolomite Small 2-Reagent chip coated with fluorine. One water inlet contained cells at the desired density to produce the desired number of cells per drop (in some embodiments, this desired number of cells per drop is one), while the second water channel contained the lysis and reaction reagent mix.

[0439] A 100 µl Hamilton Microliter syringe was used to load a 100 µl ID PEEK tubing sampling loop with two injections of approximately 100 µl each of the reaction mixture. A 100 µl Hamilton Gastight syringe was used to load approximately 110 µl of the cell suspension into a ~110 µl ID, 0.2 mm FEP loop. The loop was attached to a mechanical rotor constantly turning the loop of cells approximately once every 1-2 seconds to prevent settling and/or agglutination of the cells. The emulsion was formed by focused pressure jets of both aqueous phases passing at equal flow rates through the Dolomite 2 reagent chip with simultaneous oil flow from the two oil channels in the chip. The outer oil passages contained 0.5-5.0 (w/v) polyethylene glycol based surfactant in HFE7500 fluorocarbon oil (Novec 7500). A jet of emulsion under pressure passed the flow at a constant speed (equal to the speed of the flow containing the cells and the channels carrying the reaction mixture). The output of the emulsion chip was collected through a 12 cm PEEK tube with an inner diameter of 0.5 mm, dropwise into 0.2 ml PCR tubes in strips (Eppendorf) which were stored at approximately 0° C. in a refrigerated block.

[0440] Excess oil was removed from the bottom of each tube using a capillary micropipette. Each emulsion fraction was carefully covered with 40 μl of coating solution: 25 mm Na-EDTA, pH 8.0.

[0441] The emulsions were incubated in a thermal cycler for the transcript labeling reaction. Briefly, during a 45-minute reverse transcription (RT) step, RNA was subjected to reverse transcription at 42°C using a polyA-specific RT primer (oligo-dT primer) (SEQ ID NO: 1) and a universal adapter sequence (SEQ ID NO: 3) containing a randomized molecular barcode (see, for example, FIG. 1A) with an added matrix switch sequence. After the reverse transcription (RT) reaction, the emulsions were subjected to 40 cycles of thermal cycling (each cycle: 82°C for 10 seconds, 65°C for 25 seconds) to perform PCR amplification of the carrier barcode matrix (SEQ ID NO: 2), which was adjusted in the initial lysis and reaction mixture to a concentration of 30,000 cp-copies/µl, and creating a concentration in the final mixture of 15,000 cp/µl or ~ 1 per ~ 65 pl drop (see, for example, Fig. 1B). One end of the carrier barcode (also referred to herein as the "drop barcode") contains the Illumina read 2 ("P7") primer site (SEQ ID NO: 77), while the other end corresponds to the general sequence of the universal adapter oligonucleotide (SEQ ID NO : 4). Therefore, during template-switched PCR, the cDNA can anneal the amplified strands with the carrier barcodes and splice by overlap extension, thus resulting in full-length products containing the molecular barcodes and carrier barcode sequences.

[0442] The methods described above can be adapted to add an adapter as described in Example 5 and used for transcriptomic and target specific analysis as described in Examples 6-8.

Example 2 - Barcoding Transcripts in Emulsion Carriers Using Target Specific Primers for Single Cell Polynucleotide Sequencing.

A. Cell preparation

[0443] 50 ml of blood was drawn into Vacutainer CPT Cell Preparation Tubes for cell preparation with sodium heparin (BD), centrifuged for 20 minutes at 1800 xg, washed twice in cell preparation buffer (1x PBS supplemented with 2% fetal bovine serum and 2 mM EDTA), spun at 200 × g to remove platelets, and the resulting PBMCs were cryopreserved in RPMI-1640 (Life Technologies) + 20% fetal bovine serum + 10% DMSO at -80 °C until further operations. Before emulsion generation, PBMCs were thawed, washed twice in cell buffer: 1× Dulbecco's phosphate-buffered saline (PBS) with centrifugation (200×g for 10 min) and counted. B cells were isolated using a negative selection human B cell enrichment kit (Stem Cell Technologies). Cells were passed through a 20 µm cell filter and diluted to 6.2E+06 cells/mL (3 million B cells per experiment) or 3.1E+06 cells/mL (PGT donor and ovarian tumor experiments) in cell preparation buffer .

B. Barcoding of immune receptors in emulsion vehicles

[0444] The emulsion generation system included three Mitos P-Pumps (Dolomite Microfluidics) driven by a single air compressor, each pump equipped with a Mitos Flow Rate sensor to provide computer controlled flow of two aqueous phases and one continuous phase of Dolomite Small 2-fluorophilic oil. Reagent chip with fluorophilic coating. One water inlet contained cells at the desired density to produce the desired number of cells per drop (in some embodiments, this desired number of cells per drop is one), while the second water channel contained a mixture of the lysis and reaction reagents shown in Table E2 below, 5 U/µl MuMLV-based reverse transcriptase (Thermo Scientific) and 0.1 U/µl Herculase II PCR polymerase. A 100 µl Hamilton Microliter syringe was used to load a 100 µl ID PEEK tubing sampling loop with two injections of approximately 100 µl each of LR mix. A 100 µl Hamilton Gastight syringe was used to load approximately 110 µl of cell suspension into a ~100 µl FEP loop with an id of 0.2 mm. The emulsion was formed by focusing pressurized jets of both aqueous phases passing at equal flow rates through the Dolomite 2 reagent chip with simultaneous oil flow from the two oil channels in the chip. The emulsion leaving the chip exit channel was collected dropwise into 0.2 ml PCR tubes in strips (Eppendorf) on a chilled block, after which the excess oil was removed from the bottom of the tube with a pipette, 40 µl of coating solution (25 mM Na EDTA, pH 8.0) and then the tubes were transferred to a standard thermal cycler for the transcript labeling reaction.

Table E2. Target-Specific RT Primers IgM-RT /biotin/TGTGAGGTGGCTGCGTACTTG
(SEQ ID NO:84) IgG-RT /biotin/AGGACAGCCGGGAAGGTGT
(SEQ ID NO:85) IgD-RT /biotin/CACGCATTTGTACTCGCCTTG
(SEQ ID NO:86) IgA-RT /biotin/CTGGCTRGGTGGGAAGTTTCT
(SEQ ID NO:87) IgE-RT /biotin/GGTGGCATAGTGACCAGAGA
(SEQ ID NO:88) IgK-RT /biotin/TATTCAGCAGGCACACAACAGA
(SEQ ID NO:89) IgL-RT /biotin/AGTGTGGCCTTGTTGGCTTG
(SEQ ID NO:90) TCR-A-RT /biotin/GGGAGATCTCTGCTTCTGATG
(SEQ ID NO:91) TCR-B-RT /biotin/GGTGAATAGGCAGACAGACTTG
(SEQ ID NO:92) CD4-RT /biotin/GGCAGTCAATCCGAACACT
(SEQ ID NO:93) CD-8-RT /biotin/CTACAAAGTGGGCCCTTTCTG
(SEQ ID NO:94) IgA nested ACACGACGCTCTTCCGATCTGGCTCAGCGGGAAGACCTTG
(SEQ ID NO:44) IgE nested ACACGACGCTCTTCCGATCTGGGAAGACGGATGGGCTCTG
(SEQ ID NO:48) IgM nested ACACGACGCTCTTCCGATCTGAGACGAGGTGGAAAAGGGTTG
(SEQ ID NO:52) IgD nested ACACGAGCTCTCTTCCGATCTGGAACACATCCGGAGCCTTG
(SEQ ID NO:56) IgG nested ACACGACGCTCTTCCGATCTCCAGGGGGAAGACSGATG
(SEQ ID NO:40) IgL-nested ACACGACGCTCTTCCGATCTAGGGYGGGAACAGAGTGAC
(SEQ ID NO:60) IgK nested ACACGACGCTCTTCCGATCTGACAGATGGTGCAGCCACAG
(SEQ ID NO:64) TRA-nested ACACGACGCTCTTCCGATCTCACGGCAGGGTCAGGGTTC
(SEQ ID NO:68) TRB-nested ACACGACGCTCTTCCGATCTCGACCTCGGGTGGGAACAC
(SEQ ID NO:72) CD4 nested ACACGACGCTCTTCCGATCTTGTGGCCTTGCCGAGGGAGG
(SEQ ID NO:95) CD8 nested ACACGACGCTCTTCCGATCTTGCGGAATCCCAGAGGGCCA
(SEQ ID NO:96) C7-bc-P7 CAAGCAGAAGACGGCATACGAGATNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
(SEQ ID NO:97) C5-P5 AATGATACGGCGACCCACGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
(SEQ ID NO:98)

[0445] During a 45-minute reverse transcription (RT) of RNA at 42°C using target-specific RT primers (Table E2) with the addition of a template switch to a universal adapter sequence containing a randomized molecular barcode as previously described (Shugay , M. et al ., Towards error-free profiling of immune repertoires, Nat. Methods 11 , 653-655 (2014); Protoc 7, 813-828 (2012))) (see, for example, Fig. 1A). After reverse transcription, the emulsion carriers were subjected to 40 cycles of thermal cycling (each cycle: 82°C for 10 s, 65°C for 25 s) to perform PCR amplification of the drop barcode templates that were diluted in the initial lysis and reaction mixture up to 30,000 copies/µl, creating a concentration in the final mixture of 15,000 copies/µl or ~ 1 per ~ 65 µl drop. One end of the carrier barcode (drop barcode) contained the Illumina read 2 ("P7") primer site (SEQ ID NO: 77), while the other end corresponded to the general sequence of the universal adapter oligonucleotide (SEQ ID NO: 4) (see ., for example, Fig. 1B). Therefore, during PCR, template-switched cDNA could anneal the amplified carrier barcode strands and be spliced by overlap extension to obtain full-length products containing target sequences, molecular barcodes, and carrier barcodes.

[0446] The methods described above can be adapted to include primers for reverse transcription of a transcriptome or portion thereof, for example, by including random hexameric oligonucleotides during the reverse transcription phase. These methods can also be adapted to add an adapter, as described in Example 5, and are used for transcriptome and specific target analysis, as described in Examples 6-8.

Example 3 Barcoding method for target sequence transcripts and transcriptome in emulsion for single cell polynucleotide sequencing.

A. Cell preparation

[0447] The cryopreserved PBMC suspension is rapidly thawed and added to 10 volumes of RPMI+10% FBS at room temperature. Cells were pelleted by centrifugation at 350×g for 8 minutes and resuspended in RPMI+10% FBS to 2×10 ⁶ cells/ml. The PBMCs were placed in a tissue culture incubator for approximately 16 hours.

[0448] Rested PBMCs were co-cultured with autologous antigen presenting cells at a ratio of approximately 10:1 PBMC:APC. In this case, APCs were dendritic cells derived from autologous monocytes exposed to irradiated HSV-infected HeLa cells. The co-culture was incubated for 5 hours. Without limiting the method described here, it is believed that an incubation time of about 5 hours is sufficient for antigen-specific cells to express new mRNA in response to an antigen or in response to cytokines released into the environment by other cells.

[0449] PBMCs were removed from the co-cultivation by gentle up and down pipetting and transfer to a new tube. Cells were placed on ice and washed in CELL buffer (20 g/L fish skin gelatin (Biotium), 155 mM KCl, 0.05% sodium azide, 5 mM HEPES-Na pH 7.5) supplemented with 2 mM EDTA, then simply in CELL buffer, and finally passed through a 20 micron cell filter and resuspended in CELL buffer. Cells were counted and assessed for viability by staining with propidium iodide (acridine orange). The final cell density was adjusted to 3.5×10 ⁶ viable (propidium iodide-negative) cells/ml and stored on ice.

[0450] Just prior to emulsion formation, the cell suspension was heated and placed back on ice for 1 minute.

B. Emulsion Media Barcoding

[0451] As in the previous example, an emulsion was obtained containing prepared cells and a reaction mixture for a subsequent transcript labeling reaction by adding a molecular barcode and a cell barcode to single cell polynucleotide molecules. The reaction mixture is prepared as a 2-fold concentrate, which is mixed in a 1:1 volume ratio with the cell suspension in the process of droplet formation.

1. Preparation of the reaction mixture emulsion

[0452] A reaction mixture containing reagents and oligonucleotides was prepared as shown in Table E3 below. Oligonucleotides VB 1 (SEQ ID NO: 6), 2 (SEQ ID NO: 7), 3 (SEQ ID NO: 8) and 4 (SEQ ID NO: 9) were added as an equimolar mixture to obtain a base-shifted (stepped ) set of amplicons to increase diversity in sequencing. The reaction mixture was loaded into the reaction compartment-loop of the emulsion generating device described in the previous example.

Table E3. Reaction mixture for emulsion carriers Component Water HEPES-Na, pH 8.0 Triton X-100 (Surfact-Amps, Thermo Sci) dNTPs (each of dATP/dCTP/dTTP/dGTP) VB oligonucleotides (containing C7 adapter sequence) Reverse VB primer Direct VB primer Gene specific RT primer(s) (each) Matrix switch oligonucleotide (MB barcode) RNase inhibitor Random hexamer oligonucleotides Protease inhibitor _MgSO4 Dithiothreitol Bicarbonate of soda GTP Guanidine hydrochloride Ammonium sulfate EvaGreen Dye (Biotium) Herculase II fusion polymerase (Agilent) Maxima H - reverse transcriptase (Thermo) Oligonucleotide Sequence (5'-3') VB (media barcode) oligo 1 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT NNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A
(SEQ ID NO:6) VB (media barcode) oligo 2 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT WNNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A
(SEQ ID NO:7) VB (media barcode) oligo 3 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT NWNNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A
(SEQ ID NO:8) VB (media barcode) oligo 4 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT NNWNNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A
(SEQ ID NO:9) Direct VB primer GTGACTGGAGTTCAGACGTGTGCT
(SEQ ID NO:10) Reverse VB primer TACGTCTACGCGCTGCTCTG
(SEQ ID NO:11) IgG RT constant region primer AGGACAGCC mGmGmG AAGGTGT
(SEQ ID NO:12) IgL RT constant region primer GCTCCCGG mG T mAmG AAGTCA
(SEQ ID NO:13) IgK RT constant region primer GGCCTCTCTG mGmGmA TAGAAGT
(SEQ ID NO:14) IgM RT constant region primer TGTGAGGTGGCT mGmCmG TACTTG
(SEQ ID NO:15) IgA RT constant region primer CTGGCTRGGTG mGmGmA AGTTTCT
(SEQ ID NO:16) IgD RT constant region primer CACGCATTTGT mAmC T mC GCCTTG
(SEQ ID NO:17) IgE RT constant region primer GATGGTGGC mA T mAmG TGACCAG
(SEQ ID NO:18) TRA RT constant region primer TGTTTGAGAATCAA mAmA T mC GGTGAA
(SEQ ID NO:19) TRB RT constant region primer ACGTGGTC mGmGmG GAAGAAG
(SEQ ID NO:20) TRG RT constant region primer CAAGAAGACAAA mGmG T mA TGTTCC
(SEQ ID NO:21) TRD RT constant region primer TCTTCTTGGAT mGmAmC ACGAGA
(SEQ ID NO:22) Matrix switches, oligonucleotide (Trilink) AATACGTCCGTCAGTCGTGGATGU/(N)//(N)/T/(N)//(N)/A/(N)
//(N)/T/[(po)rG]//[(po)rG]//{3-deoxyguanosine}/
(SEQ ID NO:23) * indicates a phosphothioate bond
N=A/T/C/G
W=A/T
mA/mG/mC=2′ O-methyl A/G/C
U=2'deoxyuridine
(po)rG=riboguanosine (RNA base)

2. Creation of double barcode libraries of transcripts from single cells

[0453] An emulsion was prepared using prepared cells and a reaction mixture. The emulsion generation system included three Mitos P-Pumps (Dolomite Microfluidics) driven by a single air compressor, each pump was equipped with a Mitos Flow Rate sensor to provide computer controlled flow of two aqueous phases and one continuous fluoro oil phase Dolomite Small 2-Reagent chip with fluorine coating. One water inlet contained cells at the desired density to produce the desired number of cells per drop (in some embodiments, this desired number of cells per drop is one), while the second water channel contained a mixture of lysis and reaction reagents.

[0454] A 100 µl Hamilton Microliter syringe was used to load a 100 µl ID PEEK tubing sampling loop with two injections of approximately 100 µl each of the reaction mixture. A 100 µl Hamilton Gastight syringe was used to load approximately 110 µl of cell suspension into a ~100 µl FEP loop with an id of 0.2 mm. The loop was attached to a mechanical rotor constantly turning the cell loop about once every 1-2 seconds to prevent settling and/or agglutination of the cells. The emulsion was formed by focused pressure jets of both aqueous phases passing at equal flow rates through the Dolomite 2 reagent chip with simultaneous oil flow from the two oil channels in the chip. The outer oil passages contained 0.5-5.0 (w/v) polyethylene glycol based surfactant in HFE7500 fluorocarbon oil (Novec 7500). A jet of emulsion under pressure passed the flow at a constant speed (equal to the speed of the flow containing the cells and the channels carrying the reaction mixture). The output from the emulsion chip was collected through a 12 cm 0.5 mm ID PEEK tube, dropwise into four 0.2 ml replicate tubes in PCR strips (Eppendorf), which were stored at approximately 0° C. in a refrigerated block. Excess oil was removed from the bottom of each tube using a capillary micropipette.

[0455] The emulsions were incubated in a thermocycle to label the transcript. Briefly, the reaction mixture was pre-cooled at 4°C for 10 minutes. Then, during a 45-minute reverse transcription (RT) step, the RNA was reverse transcribed at 37°C using target-specific primers or random priming based on the binding of hexameric oligonucleotides and a universal adapter containing a randomized molecular barcode with an added template switch sequence. , thus generating cDNA molecules, each with a unique molecular identifier (barcode). After the completion of the reverse transcription reaction, the temperature was maintained at 94°C for 10 minutes. The emulsion carrier was then subjected to 50 cycles of thermal cycling (each cycle: 83° C. for 10 seconds (denaturation), 6° C. for 25 seconds (lengthening)) to amplify the carrier barcoded oligonucleotides. After VB oligo (oligonucleotide) amplification, the emulsion was subjected to 10 cycles of thermal cycling at a higher temperature (each cycle: 95°C for 10 seconds (denaturation), 63°C for 25 seconds (annealing), 72°C for 2 minutes 20 seconds (elongation) After the thermal cycling cycles were completed, the emulsion was held at 4°C.

Example 4 Purification of cDNA with two barcodes

[0456] For each emulsion fraction tube, after barcoding the cDNA transcripts with the two barcodes obtained in the examples above, the emulsion was broken (after PCR) by mixing with an equal volume of 1:1 (v:v) perfluorooctanol: FC- 40 and EDTA was added at a final concentration of 5 mM to prevent DNA polymerization. Approximately 0.1 volume of Qiagen Protease was added and the broken emulsion was incubated at 50°C for 10-15 minutes followed by heat inactivation of the protease by incubating the tubes at 95°C for 3 minutes. The tube was quickly centrifuged and the upper aqueous phase was transferred to a new tube.

[0457] Double-barcoded cDNA was concentrated and desalted by 1.8 volume AMPure XP purification (Beckman Coulter) as recommended by the manufacturer. cDNA was eluted from the beads and denatured by adding 8 μl of 0.1 M sodium hydroxide + 1 mm EDTA and heating to 50°C for 3 minutes. If full-length products contained biotin due to 5'-biotinylation of the RT primer, such full-length products were separated from the rest of the PCR products with a barcode in the drop by purification on streptavidin beads.

[0458] Following the addition of 2 μl of 6X DNA sample gel dye (New England Biolabs), denatured single-stranded cDNA was run/separated on a 1.5% (w/v) agarose gel in 30 mM NaOH + 1 mM EDTA at 5 V/cm for 35 minutes. After the pH of the gel was neutralized, the gel containing cDNA in the size range corresponding to 100-1000 bp was excised and the cDNA was purified from agarose using a DNA recovery kit (e.g., Zymoclean™ Gel DNA Recovery Kit, Zymo Research.) eluting in 20 µl 10 mM Tris-Cl, pH 8.5+0.05% TWEEN® 20. Next, cDNA was desalted by adding 1.8 volumes of AMPure XP beads, eluted in 10.5 µl 10 mM Tris-Cl, pH 8.0+ 0.05% TWEEN® 20 when heated at 95°C for 10s and deposited by placing on ice.

Example 5 - Ligation of the 3' adapter sequence with cDNA transcripts with two barcodes

[0459] An adapter sequence containing a known priming site was added to the dual barcode cDNA transcripts generated as described in Example 3 after purification as described in Example 4. The addition of an adapter sequence allows amplification, cloning or sequencing such as next generation sequencing, all transcripts with a known primer. Some adapter sequences, such as the exemplary P5 adapter sequence used herein, are known and commonly used for sequencing.

1. Methods for adding a 3' adapter sequence

[0460] Several methods have been used to add an adapter sequence to the 3' end of single-stranded, unknown cDNA sequences with two barcodes. Ligases such as Thermostable App ligase (NEB) and CircLigase II (Epicenter), which ligate the ssDNA adapter, have been used to add the adapter sequence to the 3' end of double-barcoded cDNA transcripts. A commercial kit (Swift Biosciences Accel-NGS 1S DNA Kit) providing enzymatic addition of non-template nucleotides to the 3' end of the cDNA was also used to add the adapter sequence. In addition, methods have been used where a degenerate hinge splint oligonucleotide is annealed with an adapter projecting over the 3' end of the cDNA with degenerate overhangs up to 6 nucleotides (ie, NNNNNN; SEQ ID NO: 24). For degenerate overhangs, the sequence NNNNNN (SEQ ID NO: 24) has proven to be the most successful in the protocols tested.

2. Addition of 3' adapter sequence to double-barcoded cDNA using a degenerate 6 nt overhang splint

[0461] A duplex molecule adapter-hinge (splint) nucleotide was obtained by mixing oligonucleotides containing a short priming sequence P5, /5Phos/ AGATCGGAAGAGCGTCGTGT /3AmMO/ (SEQ ID NO: 25) and a hinge (splint) oligonucleotide designated as ACACGACGCTCTTCCGATCT NNNNNN /3AmMO / (SEQ ID NO:26) in a ratio of 1.2: 1. Annealing buffer was added to 30 mm HEPES-Na pH 7.5, 0.1 M KCl. The solution was heated at 85°C for 2 minutes in a thermal cycler and cooled to 37°C at a rate of 0.1°C/sec.

[0462] The double-barcoded cDNA transcripts isolated from part A above were then mixed with the hinge oligonucleotide adapter solution. The adapters were then ligated to the cDNA transcripts by adding an equal volume of Blunt/TA Ligase Master Mix (New England Biolabs) to the mixture and incubating at room temperature. Excess adapter DNA was removed by purifying the mixture with AMPure XP and eluting the DNA in 10 mM Tris-Cl, pH 8.0 + 0.05% TWEEN® 20.

Example 6 - PCR amplification and transcriptome library sequencing

A. Amplification of a polynucleotide library

[0463] Purified sequences labeled with a double barcode and labeled with a universal adapter were amplified by PCR for 8 cycles using a forward primer complementary to the C7 universal adapter sequence (SEQ ID NO: 28) located 5' from the double barcode and the coding sequence of the transcript (5' end of the cDNA transcript) and the reverse primer complementary to the P5 universal adapter sequence (SEQ ID NO: 27) located at the 3' position from the double barcode and the coding sequence of the transcript (3' end of the cDNA transcript) (PCR0).

[0464] The PCR0 reaction mixture (containing cDNA ligated to adapter and (obtained in Example 5), DNA polymerase, C7 forward primer, P5 reverse primer, dNTP and reaction buffer) was first denatured at 98°C for 1 min, and then 8 cycles of thermal cycling were carried out (each cycle: 98°C for 10 s, 69°C for 20 s, 72°C for 10 s) with a final elongation time of 2 min at 72°C. After completion of PCR, the mixture was kept at 4°C.

[0465] Each cDNA sequence resulting from PCR0 contained a C7 adapter sequence, a carrier barcode sequence (to identify the host cell), a molecular barcode (to identify the transcript), a transcript, and a P5 adapter sequence. The amplified library (PCR0 product) was then purified using AMPure XP beads and eluted in 10 mM Tris-Cl, pH 8.0 + 0.05% TWEEN® 20.

[0466] The purified transcriptome library was then used to sequence one or more of the list: one or more full-length target genes, such as immune receptor, the transcriptome of all cells in the emulsion, and/or the transcriptome of one or more selected cells in the emulsion, as described below.

B. Sequencing of target genes

1. PCR1: amplification of the target gene(s)

[0467] To amplify the selected target gene, the PCR0 product was amplified using the universal forward primer used for PCR0 (primer C7-index-P7; SEQ ID NO: 28) and a reverse primer specific for the desired target sequence(s). (s) (as, for example, specific immunoglobulin receptors of the T cell receptor). Representative target-specific primers used for the PCR1 reaction are shown in Table E4 below.

Table E4. Exemplary target-specific reverse primer sequences Target Primer sequence 5' to 3' IgG constant region AAGTAGTCCTTGACAGGCAGC
(SEQ ID NO:29) IgL constant region GGCTTGAAGCTCCTCAGAGGA
(SEQ ID NO:30) IgK constant region AGGCACCACAACAGAGGCAGTTC
(SEQ ID NO:31) IgM constant region CGACGGGGAATTCTCACAGGAG
(SEQ ID NO:32) IgD constant region TGTCTGCACCCTGATATGATGG
(SEQ ID NO:33) IgA constant region 1 GGGTGCTGCAGAGGCTCAG
(SEQ ID NO:34) IgA constant region 2 GGGTGCTGTCGAGGCTCAG
(SEQ ID NO:35) IgE constant region GGAATGTTTTTGCAGCAGCGGG
(SEQ ID NO:36) TRA constant region AGTCTCTCAGCTGGTACACGG
(SEQ ID NO:37) TRB constant region ATGGCTCAAACACAGCGACCTC
(SEQ ID NO:38)

[0468] The PCR1 reaction mixture (containing the PCR0 product (obtained in part A above), DNA polymerase, C7-index-P7 forward primer, target-specific reverse primer, dNTPs, and reaction buffer) was initially denatured at 98°C. for 1 minute followed by 10 cycles of thermocycling (each cycle: 98°C for 10 s, 61°C for 20 s, 72°C for 20 s) with a final elongation time of 2 min at 72°C. After completion of PCR1, the mixture was kept at 4°C. The PCR1 product (target gene sequence(s)) was purified with AMPure XP beads and eluted in 10 mM Tris-Cl, pH 8.0 + 0.05% TWEEN® 20.

2. PCR2: amplification of the target gene with the addition of the 3'-sequence of the adapter sequence

[0469] The purified PCR1 product was amplified using the C7 forward primer (SEQ ID NO: 39) and a target specific reverse primer containing the P5 short universal priming sequence. Representative primers used for the PCR2 reaction are shown in Table E5 below.

Table E5. Exemplary target-specific reverse primer sequences Target Primer sequence 5' to 3' IgG constant AAGTAGTCCTTGACAGGCAGC
(SEQ ID NO:29) IgL constant GGCTTGAAGCTCCTCAGAGGA
(SEQ ID NO:30) IgK constant AGGCACCACAACAGAGGCAGTTC
(SEQ ID NO:31) IgM constant CGACGGGGAATTCTCACAGGAG
(SEQ ID NO:32) IgD constant TGTCTGCACCCTGATATGATGG
(SEQ ID NO:33) IgA constant 1 GGGTGCTGCAGAGGCTCAG
(SEQ ID NO:34) IgA constant 2 GGGTGCTGTCGAGGCTCAG
(SEQ ID NO:35) IgE constant GGAATGTTTTTGCAGCAGCGGG
(SEQ ID NO:36) TRA constant AGTCTCTCAGCTGGTACACGG
(SEQ ID NO:37) TRB constant ATGGCTCAAACACAGCGACCTC
(SEQ ID NO:38)

[0470] PCR2 reaction mixture (containing the PCR1 product (obtained in part B1 above), DNA polymerase, C7 forward primer (SEQ ID NO: 39), target-specific reverse P5 short (short P5) primer, dNTP and reaction buffer) initially denatured at 98°C for 1 minute, followed by 6 cycles of thermal cycling (each cycle: 98°C for 10 s, 65°C for 20 s, 72°C for 20 s) and a final elongation time of 2 min at 72°C. After completion of PCR2, the mixture was kept at 4°C. The PCR2 product (target gene sequence(s)) was purified with AMPure XP beads and eluted with 10 mM Tris-Cl, pH 8.0 + 0.05% TWEEN® 20.

3. Quantitative PCR (qPCR3) and target gene sequencing

[0471] The purified PCR2 or PCR0 product was used for quantitative PCR (qPCR) to determine the number of amplification cycles to reach the qPCR3 endpoint. Pre-amplified adapter-ligated material downstream of PCR0 or full-length IG or TR PCR2 products were amplified with primers C7 (forward; SEQ ID NO: 39) and C5-P5 (reverse; SEQ ID NO: 76).

[0472] Briefly, the qPCR3 reaction mixture (containing the PCR0 or PCR2 product (obtained in parts A or B1 above, respectively), DNA polymerase, C7 forward primer (SEQ ID NO: 39), C5-P5 reverse primer (SEQ ID NO : 76), dNTP, EvaGreen and reaction buffer) were first denatured at 98°C for 1 minute, and then 3 thermocycling cycles were performed (each cycle: 98°C for 10 s, 60°C for 20 s, 72° C for 20 s), followed by 30 cycles of a second thermocycling cycle (each cycle: 98°C for 10 s, 70°C for 20 s, 72°C for 20 s). The qPCR intensity plot was checked to determine the amplification cycle at which the fluorescence intensity was maximum, but the DNA amplification had not yet ended. This served to determine the number of cycles to reach the qPCR3 endpoint.

[0473] After determining the number of PCR cycles required to amplify each library to exponential phase, the same PCR was repeated non-quantitatively to the desired number of cycles and the PCR products were purified with 1 AMPure XP and eluted in 10 mM Tris-Cl., pH 8, 0+0.05% TWEEN® 20. Optionally, qPCR3 (qPCR3) results were normalized with DASH (Gu et al., Genome Biology 2016, 17:41). Results were analyzed with the Agilent Tapestation D1000, quantified with the KAPA NGS Quant Kit for Illumina, and sequenced with the Illumina NextSeq high output 75-cycle kit (e.g., 32 Read 1 cycles, 6 Index Read I7 cycles, 54 Read cycles). 2) for a ligated adapter library or Illumina MiSeq V3 600-cycle kit (e.g., 325 Read 1 cycles, 6 Index Read I7 cycles, 300 Read 2 cycles) for full size IG and TR libraries. In some cases, the NextSeq sequencer ran 56 Read 1 cycles, 6 Index Read I7 cycles, and 33 Read 2 cycles.

C. Sequencing of transcriptomes from all cells

[0474] To create a transcriptome library from all cells, PCR1, PCR2, and qPCR3 (qPCR3) reactions are performed as described above, except for those where there is a replacement of target-specific reverse primers with universal reverse primers directed to the 3' adapter sequence ( eg, SEQ ID NOS: 27 and 76) for use in combination with universal forward primers (eg, SEQ ID NOS: 28 and 39) for amplification. Thus, universal forward and reverse primers can be used to sequence all transcripts of all cells in the emulsion.

D. Transcriptome sequencing of selected cells

[0475] To create a transcriptome library of selected cells, PCR1, PCR2, and qPCR3 (qPCR3) reactions are performed as described above, except when forward primers are used that are complementary to the carrier barcode (VB) of the desired cell or cells, such as cell or cells containing the Ig molecule or TCR of interest sequenced in part A above, and universal reverse primers directed to the 3' adapter sequence (eg, SEQ ID NOS: 27 and 76).

Example 7 - Sequence Data Analysis

[0476] Illumina MiSeq reads were processed to generate full-length consensus sequences of molecules and mRNA droplets, annotated with IgBLAST and IMGT/HighV-QUEST, and processed with user programs and the Change-0 package to generate statistics and numbers. MiSeq reads were demultiplexed using the Illumina software. Results with reading quality below Phred 5 were masked with Ns. Isotype-specific primers, carrier barcodes (VB), molecular barcodes (MB) and adapter sequences were identified in the amplicon and excluded using pRESTO MaskPrimers-cut with a maximum error of 0.2.

A. Data Analysis of Selected Immune Receptor Sequence

[0477] In one example, full length target immune receptor sequences were generated and sequenced. The Read 1 consensus sequence and the Read 2 consensus sequence were generated separately for each mRNA from reads grouped by a unique molecular identifier (UMI) including VB and MB together, which are PCR replicas arising from the same parent mRNA molecule. Read group UMIs were aligned using MUSCLE and pRESTO was used to build consensus sequences with the following parameters: maxdiv=0.1; bf PRIMER; prfreq=0.6; maxmiss=0.5; q=5; > 60% sequence match of the selected PCR primers for the read group; maximum nucleotide diversity=0.1; using the majority rule on indel positions; and masking alignment for columns with low quality consensus sequence. The paired terminal consensus sequences were then aligned in two rounds. First, the ungapped alignment of the ends of the consensus sequences of each read pair was optimized using the Z-score approximation and estimated using the binomial p (p-value), as provided in pRESTO AssemblePairs-align with the following parameters: minimum length=8; alpha l×10 ⁵ ; and maximum error=0.3. For pairs of reads that could not be stitched in this way, stitching was attempted using human BCR and TCR germline V-exons to protect each read before subsequent stitching or gap alignment using pRESTO reference parameters. AssemblePairs: minimum identity=0.5; e value l×10 ⁵ .

1. V D J segment annotation and isotype confirmation

[0478] IgBLAST, Change-0, and user programs were used to identify gene lineage, V(D)J germline, truncate mRNA sequences to the V(D)J region, identify CDR3 regions, and enumerate mutations in V germline nucleotide sequences. IgBLAST counts Ns as mismatches, but mRNA sequences with more than 6 mismatches (Ns) in the V region were selected for mutation analysis and accurate pairing analysis between fractions. For IG heavy chains, isotype identity was confirmed by matching non-primer C regions (constant region exons) to expected sequences using pRESTO MaskPrimers-Score parameters: start=0; maximum error=0.2. Amplicons with discordant primer/non-primer signals in the C-region were discarded, except for two primer/non-primer combinations where, in the presence of certain interaction interference, all were resolved by visual inspection.

2. Grouping of V (D) J sequences into clonal lines

[0479] V(D)J sequences were grouped into clones using single-stranded clustering with interclonal distance taken into account. Clustering was performed using the Change-0 DefineClones-by package with group parameters: model=mln; gene=first; dist=4.0; norm=none. First, the consensus sequences of all functional Ig VH droplet chains were pooled into samples having VJ junction so that sequences possibly resulting from the same initial recombination event were pooled together (based on Ig VH gene best match, best match Ig JH gene and junction boundary length as determined by IMGT/HighV-QUEST). The interclonal distance threshold was chosen by generating a histogram of nearest neighbor distances within each VH Ig sample using the distToNearest function of Change-0's shm package and visually checking the histogram to determine natural distance (at the bimodal histogram trough). Clonal light chain clusters were determined using the same distance model and threshold.

3. Creation of a sample of drops, calculation of the pairing accuracy

[0480] The reliability of pairing (strong-loose) was assessed in two independent ways: based on the matching of mRNA sequences within the droplet and the matching of sequences between replicas. The mRNA match within the droplet was determined as the mean pairwise nucleotide difference (Nei's pi < 0.02) of the V (D) J sequences within the locus. The mRNA sequences were trimmed to V(D)J nucleotide coding sequences using IgBLAST annotations. Within each drop, all working mRNA sequences were grouped according to the V locus. Within each group, multiple sequences were aligned using MUSCLE as presented in pRESTO AlignSets using the default settings. Droplet consensus strands were built from multiple mRNAs per locus using the pRESTO parameters: BuildConsensus.py; maxdiv=0.2; maximum miss=0.5. Drops taken at random were used to select a diversity constraint pi < 0.02. In randomized droplets, less than 0.01% of the heavy chain loci (and <0.2% of the light chain loci) met this criterion. Droplets containing multiple cells or immune receptors were separated for further precise analysis.

является числом 1 штрих-кодов носителей d со спаренными «тяжелым» клоном h и «легким» клоном 1, находящихся в физической фракции f. Средняя (квадратичная) точность спаривания для каждого эксперимента оценивается путем усреднения по тяжелым клонам h и по всем парам фракций (f, g), и соответствию спаренных «легких» клонов (1, k): [0481] Mating accuracy was calculated based on the observation of the same pair in the same clone in several replicas (separate emulsion experiments), focusing on those VDJ clusters that are most likely to contain only one line, i.e., originating from the same V(D)J by VJ permutation followed by expansion. Similar VDJ rearrangements can occur over several independent periods, resulting in a rearrangement of the same V(D)J heavy chain naturally associated with many different VJ light chain rearrangements. Because the sparse V(D)J permutations could provide a more accurate measure of the technical accuracy achieved by the methods described here, the long heavy CDR3 (CDR3H) was not the focus of this analysis (as a proxy for the sparse V(D)J permutations). Sequences with >6N were also removed to increase the certainty of clonal affiliation. Mating accuracy increased with CDR 3 H length to over 96% for the longest clone quartile observed among the fractions (2,604 clones with ≥54 nt bond length). Since the probability of matching park-clones is a function of the overall probability of true pairs, in two independent experiments, the matching accuracy was estimated as the square root of the pairwise matching across replicas, calculated as follows:

is the number 1 of barcodes of carriers d with paired "heavy" clone h and "light" clone 1, located in the physical fraction f. The average (quadratic) pairing accuracy for each experiment is estimated by averaging over heavy clones h and over all pairs of fractions (f, g), and the correspondence of paired "light" clones (1, k):

[0482] Therefore, the average accuracy of each experiment (up to the variance of accuracy between experiments) was 96.1% in accordance with this illustrative experiment.

B. Analysis of transcriptome sequence data

[0483] For the obtained transcriptome sequence data, reads containing the same VB were folded and the sequences were matched against a reference human genome for transcript identification (HiSAT2). Output file alignment was performed using samtools, and reads were assigned to transcripts according to their genomic location.

[0484] For each mapping of the VB genome, the reads were folded by MB. The matrix was constructed from MB calculations mapped to each individual reference gene per drop. These data were then combined with target data, eg immune receptor sequence data processed as described above in Part A. The data for each VB (droplet) was annotated with gene number and receptor information. The pooled datasets were then analyzed to examine the RNA sequence profile (scRNAseq) of a single cell. Size reduction, clustering, and visualization were performed using t-SNE, Seurat, ZIFA, PCA, LDA, and other typical programs.

Example 8 Illustrative High Throughput Sequenced Transcriptome Sequence Data Analysis and Plotting on Multiple Single Cells

[0485] Approximately 7,000 PBMCs were isolated, transcriptomes, and full-length BCR and TCR receptors were sequenced as typically described in Examples 4A and 4B above.

[0486] Prior to Illumina NextSeq analysis, the emulsion carrier transcriptome sequencing libraries prepared as described in Example 4B were analyzed using a D1000 DNA tapestation. The transcriptome was represented by sequences of approximately 170-900 bp in size. Full length TCR sequences sequenced by the targeted sequencing described in Example 4A were also analyzed with the D1000 DNA tapestation prior to NextSeq analysis, which showed TCR alpha and beta peaks at 628 and 664 bp. respectively.

[0487] Following NextSeq analysis, all droplet cell profiles with >1000 reads (n=6.707) were analyzed using t-distribution stochastic neighbor nesting (t-SNE) and Seurat clustering. Multivariate single cell transcriptome data were visualized using t-SNE plots and cells were color-coded based on Seurat cell clustering with a similar transcription profile (Fig. 3A) or according to the nature of the sequenced immune receptor (i.e., BCR (red/ standard grey) or TCR (green/dark grey)) (Fig. 3B), to demonstrate clustering of cells with similar phenotypes.

[0488] Transcriptome data for representative genes in sequenced cells were analyzed and sequence information with the same carrier barcode as the sequenced immune receptor was interpreted as being from the same cell. Transcriptome data for typical genes in a single cell transcriptome are color-coded on a heat map of expression levels and shown for Toll-like receptor 7 (TLR7; Fig. 4A), CD3 glycoprotein epsilon chain on the surface of T cells (CD3E; Fig. 4B), Natural killer cell bead protein 7 (NKG7; Fig. 4C), MRC1 C-type 1 mannose receptor (MRC1; Fig. 4D).

[0489] These results indicate that genome-wide RNA expression profiles can be determined along with high throughput immune receptors, since genetic markers associated with T cells (CD3E) or B cells (TLR7) tend to cluster in cells expressing full-length TCRs or BCRs, respectively. Similarly, genetic markers not associated with T cells or B cells, such as the typical NK cell marker NKG7 or the typical monocyte marker MRC1, do not appear to cluster in cells with full length TCR or BCR immune receptors.

[0490] The specific disclosed embodiments are not intended to limit the scope of the present invention, but are provided, for example, to illustrate various aspects of the invention. Various modifications of the described compositions and methods will become apparent from the description and ideas given here. Such variations may be practiced without deviating from the true scope and spirit of the disclosure and should be considered to fall within the scope of this disclosure.

SEQUENCES

# SUBSEQUENCE ANNOTATION 1 /5BiosG//iSpl8/TTT TTT TTT TTT TTT TTT TTT TTT T V N 5'biotin oligo-dT cross-linked reverse transcription primer 2 ATCCATCCACGACTGACGGACGTATTAAANNNNWNNNNWNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCACC matrix with carrier barcode, oligo 3 AATACGTCCGTCAGTCGTGGATGNNTNNANNTrGrGG matrix switching, oligo 4 CATCCACGACTGACGGACGTATT direct primer with carrier barcode/universal adapter, oligonucleotide 5 GTGACTGGAGTTCAGACGTGTGCT reverse primer with carrier barcode 6 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT NNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A media barcode, oligo 1 7 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT WNNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A media barcode, oligo 2 8 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT NWNNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A media barcode, oligo 3 9 T*A*C*G*TCTACGCGCTGCTCTG CCACGACTGACGGACGTATT NNWNNNNWNNNNWNNNNAGATCGGAAG AGCACACGTCTGAACTCCA*G*T*C*A media barcode, oligo 4 10 GTGACTGGAGTTCAGACGTGTGCT direct primer with carrier barcode eleven TACGTCTACGCGCTGCTCTG reverse primer with carrier barcode 12 AGGACAGCC mGmGmG AAGGTGT IgG RT primer 13 GCTCCCGG mG T mAmG AAGTCA IgL RT constant region primer 14 GGCCTCTCTG mGmGmA TAGAAGT IgK RT constant region primer 15 TGTGAGGTGGCT mGmCmG TACTTG IgM RT constant region primer 16 CTGGCTRGGTG mGmGmA AGTTTCT IgA RT constant region primer 17 CACGCATTTGT mAmC T mC GCCTTG IgD RT constant region primer 18 GATGGTGGC mA T mAmG TGACCAG IgE RT constant region primer 19 TGTTTGAGAATCAA mAmA T mC GGTGAA TRA RT constant region primer 20 ACGTGGTC mGmGmG GAAGAAG TRB RT constant region primer 21 CAAGAAGACAAA mGmG T mA TGTTCC TRG RT constant region primer 22 TCTTCTTGGAT mGmAmC ACGAGA TRD RT constant region primer 23 AATACGTCCGTCAGTCGTGGATGU/(N)//(N)/T/(N)//(N)/A/(N)//(N)/T/[(po)rG]//[(po)rG]/ /{3-deoxyguanosine}/ Matrix switching, oligo (Trilink) 24 NNNNNN degenerate ledges 25 /5Phos/ AGATCGGAAGAGCGTCGTGT /3AmMO short P5 (short P5) priming sequence 26 ACACGACGCTCTTCCGATCT NNNNNN /3AmMO/ hinged (splint) oligonucleotide 27 ACACGACGCTCTTCCGATCT short P5 (short P5) reverse primer 28 CAAGCAGAAGACGGCATACGAGAT[NNNNNN]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT C7-index-P7 direct primer 29 AAGTAGTCCTTGACAGGCAGC IgG reverse primer constant region sequence thirty GGCTTGAAGCTCCTCAGAGGA IgL reverse primer constant region sequence 31 AGGCACCACAACAGAGGCAGTTC IgK reverse primer constant region sequence 32 CGACGGGGAATTCTCACAGGAG IgM reverse primer constant region sequence 33 TGTCTGCACCCTGATATGATGG IgD reverse primer constant region sequence 34 GGGTGCTGCAGAGGCTCAG IgA reverse primer constant region 1 sequence 35 GGGTGCTGTCGAGGCTCAG IgA reverse primer constant region 2 sequence 36 GGAATGTTTTTGCAGCAGCGGG IgE reverse primer constant region sequence 37 AGTCTCTCAGCTGGTACACGG TRA reverse primer constant region sequence 38 ATGGCTCAAACACAGCGACCTC TRB reverse primer constant region sequence 39 CAAGCAGAAGACGGCATACGAGAT C7 40 ACACGACGCTCTTCCGATCTCCAGGGGGAAGACSGATG IgG target-specific reverse primer sequence with tethered adapter 41 ACACGACGCTCTTCCGATCTNCCAGGGGGAAGACSGATG IgG target-specific reverse primer sequence with tethered adapter 42 ACACGACGCTCTTCCGATCTNNCCAGGGGGAAGACSGATG IgG target-specific reverse primer sequence with tethered adapter 43 ACACGACGCTCTTCCGATCTNNNCCAGGGGGAAGACSGATG IgG target-specific reverse primer sequence with tethered adapter 44 ACACGACGCTCTTCCGATCTGGCTCAGCGGGAAGACCTTG IgA target-specific reverse primer sequence with tethered adapter 45 ACACGACGCTCTTCCGATCTNGGCTCAGCGGGAAGACCTTG IgA target-specific reverse primer sequence with tethered adapter 46 ACACGACGCTCTTCCGATCTNNGGCTCAGCGGGAAGACCTTG IgA target-specific reverse primer sequence with tethered adapter 47 ACACGACGCTCTTCCGATCTNNNGGCTCAGCGGGAAGACCTTG IgA target-specific reverse primer sequence with tethered adapter 48 ACACGACGCTCTTCCGATCTGGGAAGACGGATGGGCTCTG IgE target-specific reverse primer sequence with tethered adapter 49 ACACGACGCTCTTCCGATCTNGGGAAGACGGATGGGCTCTG IgE target-specific reverse primer sequence with tethered adapter 50 ACACGAGCTCTCTTCCGATCTNNGGGAAGACGGATGGGCTCTG IgE target-specific reverse primer sequence with tethered adapter 51 ACACGACGCTCTTCCGATCTNNNGGGAAGACGGATGGGCTCTG IgE target-specific reverse primer sequence with tethered adapter 52 ACACGACGCTCTTCCGATCTGAGACGAGGTGGAAAAGGGTTG IgM target-specific reverse primer sequence with tethered adapter 53 ACACGACGCTCTTCCGATCTNGAGACGAGGTGGAAAAGGGTTG IgM target-specific reverse primer sequence with tethered adapter 54 ACACGACGCTCTTCCGATCTNNGAGACGAGGTGGAAAAGGGTTG IgM target-specific reverse primer sequence with tethered adapter 55 ACACGACGCTCTTCCGATCTNNNGAGACGAGGTGGAAAAGGGTTG IgM target-specific reverse primer sequence with tethered adapter 56 ACACGAGCCTTTCCGATCTGGAACACATCCGGAGCCTTG IgD target-specific reverse primer sequence with tethered adapter 57 ACACGACGCTCTTCCGATCTNGGAACACATCCGGAGCCTTG IgD target-specific reverse primer sequence with tethered adapter 58 ACACGAGCTCTCTTCCGATCTNNGGAACACATCCGGAGCCTTG IgD target-specific reverse primer sequence with tethered adapter 59 ACACGACGCTCTTCCGATCTNNNGGAACACATCCGGAGCCTTG IgD target-specific reverse primer sequence with tethered adapter 60 ACACGACGCTCTTCCGATCTAGGGYGGGAACAGAGTGAC IgL target-specific reverse primer sequence with tethered adapter 61 ACACGACGCTCTTCCGATCTNAGGGYGGGAACAGAGTGAC IgL target-specific reverse primer sequence with tethered adapter 62 ACACGACGCTCTTCCGATCTNNAGGGYGGGAACAGAGTGAC IgL target-specific reverse primer sequence with tethered adapter 63 ACACGACGCTCTTCCGATCTNNNAGGGYGGGAACAGAGTGAC IgL target-specific reverse primer sequence with tethered adapter 64 ACACGACGCTCTTCCGATCTGACAGATGGTGCAGCCACAG IgK target-specific reverse primer sequence with tethered adapter 65 ACACGACGCTCTTCCGATCTNGACAGATGGTGCAGCCACAG IgK target-specific reverse primer sequence with tethered adapter 66 ACACGACGCTCTTCCGATCTNNGACAGATGGTGCAGCCACAG IgK target-specific reverse primer sequence with tethered adapter 67 ACACGACGCTCTTCCGATCTNNNGACAGATGGTGCAGCCACAG IgK target-specific reverse primer sequence with tethered adapter 68 ACACGACGCTCTTCCGATCTCACGGCAGGGTCAGGGTTC TRA target-specific reverse primer sequence with tethered adapter 69 ACACGACGCTCTTCCGATCTNCACGGCAGGGTCAGGGTTC TRA target-specific reverse primer sequence with tethered adapter 70 ACACGACGCTCTTCCGATCTNNCACGGCAGGGTCAGGGTTC TRA target-specific reverse primer sequence with tethered adapter 71 ACACGACGCTCTTCCGATCTNNNCACGGCAGGGTCAGGGTTC TRA target-specific reverse primer sequence with tethered adapter 72 ACACGACGCTCTTCCGATCTCGACCTCGGGTGGGAACAC TRB target-specific reverse primer sequence with tethered adapter 73 ACACGACGCTCTTCCGATCTNCGACCTCGGGTGGGAACAC TRB target-specific reverse primer sequence with tethered adapter 74 ACACGACGCTCTTCCGATCTNNCGACCTCGGGTGGGAACAC TRB target-specific reverse primer sequence with tethered adapter 75 ACACGACGCTCTTCCGATCTNNNCGACCTCGGGTGGGAACAC TRB target-specific reverse primer sequence with tethered adapter 76 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC C5-P5 reverse primer 77 AGATCGGAAGAGCACACGTCTGAACTCCA P7 priming site (C7) 78 AGATCGGAAGAGCGTCGTGTAGGGAAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT P5 priming site 79 ACACGACGCTCTTCCGATCTNNNNNN Universal adapter; where N is any nucleotide 80 NNNNWNNNNWNNNN Universal adapter; where N is any nucleotide and W is adenine or thymine 81 WNNNNWNNNNWNNNN Universal adapter; where N is any nucleotide and W is adenine or thymine 82 NWNNNWNNNNWNNNN Universal adapter; where N is any nucleotide and W is adenine or thymine 83 NNWNNNNWNNNNWNNNN Universal adapter; where N is any nucleotide and W is adenine or thymine 84 /biotin/TGTGAGGTGGCTGCGTACTTG IgM-RT primer 85 /biotin/AGGACAGCCGGGAAGGTGT IgG-RT primer 86 /biotin/CACGCATTTGTACTCGCCTTG IgD-RT primer 87 /biotin/CTGGCTRGGTGGGAAGTTTCT IgA-RT primer 88 /biotin/GGTGGCATAGTGACCAGAGA IgE-RT primer 89 /biotin/TATTCAGCAGGCACACAACAGA IgK-RT primer 90 /biotin/AGTGTGGCCTTGTTGGCTTG IgL-RT primer 91 /biotin/GGGAGATCTCTGCTTCTGATG TCR-A-RT primer 92 /biotin/GGTGAATAGGCAGACAGACTTG TCR-B-RT primer 93 /biotin/GGCAGTCAATCCGAACACT CD4-RT primer 94 /biotin/CTACAAAGTGGGCCCTTTCTG CD-8-RT primer 95 ACACGACGCTCTTCCGATCTTGTGGCCTTGCCGAGGGAGG CD4 nested primer 96 ACACGACGCTCTTCCGATCTTGCGGAATCCCAGAGGGCCA CD8 nested primer 97 CAAGCAGAAGACGGCATACGAGATNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT C7-bc-P7 primer 98 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT C5-P5 primer 99 NNNNWNNNNWNNNNWNNNNW Media Barcode Sequence 100 NNNNWISCNNNNWISCNNNN Media Barcode Sequence

--->

SEQUENCE LIST

<110> Godfless, Stephen Jacob

Briggs, Adrian Wrangham

Chari, Rajagopal

Jiang, Yu

House, Ronald

Vigno, Francois

<120> HIGH CAPACITY SEQUENCE

AND ANALYSIS OF THE TRANSCRIPTOME

<130> 735042011740

<140> destination not yet defined

<141> At the same time as

<150> US 62/511,949

<151> 2017-05-26

<160> 100

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> 5'Biotin-labeled oligo(dT)-linked primer for reverse transcription

<220>

<221> other_characteristic

<222> 1

<223> 5' biotin modification with 18-carbon spacer

<220>

<221> other_characteristic

<222> 27

<223> n = A, T, C or G

<400> 1

tttttttttt tttttttttt tttttvn 27

<210> 2

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> carrier barcode matrix, oligo

<220>

<221> other_characteristic

<222> 30, 31, 32, 33, 35, 36, 37, 38, 40, 41, 42, 43

<223> n = A, T, C or G

<400> 2

atccatccac gactgacgga cgtattaaan nnnwnnnnwn nnnagatcgg aagagcacac 60

gtctgaactc cagtcacc 78

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> matrix switching, oligo

<220>

<221> other_characteristic

<222> 33

<223> riboguanosine

<220>

<221> other_characteristic

<222> 34

<223> riboguanosine

<220>

<221> other_characteristic

<222> 24, 25, 27, 28, 30, 31

<223> n = A, T, C or G

<400> 3

aatacgtccg tcagtcgtgg atgnntnnan ntggg 35

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> direct primer/universal adapter with media barcode,

oligonucleotide

<400> 4

catccacgac tgacggacgt att 23

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer with carrier barcode

<400> 5

gtgactggag ttcagacgtg tgct 24

<210> 6

<211> 87

<212> DNA

<213> Artificial sequence

<220>

<223> media barcode, oligo 1

<220>

<221> other_characteristic

<222> (1)...(5)

<223> phosphorothioate bond

<220>

<221> other_characteristic

<222> (83)...(87)

<223> phosphorothioate bond

<220>

<221> other_characteristic

<222> 41, 42, 43, 44, 46, 47, 48, 49, 51, 52, 53, 54

<223> n = A, T, C or G

<400> 6

tacgtctacg cgctgctctg ccacgactga cggacgtatt nnnnwnnnnw nnnnagatcg 60

gaagagcaca cgtctgaact ccagtca 87

<210> 7

<211> 88

<212> DNA

<213> Artificial sequence

<220>

<223> media barcode, oligo 2

<220>

<221> other_characteristic

<222> (1)...(5)

<223> phosphorothioate bond

<220>

<221> other_characteristic

<222> (84)...(88)

<223> phosphorothioate bond

<220>

<221> other_characteristic

<222> 42, 43, 44, 45, 47, 48, 49, 50, 52, 53, 54, 55

<223> n = A, T, C or G

<400> 7

tacgtctacg cgctgctctg ccacgactga cggacgtatt wnnnnwnnnn wnnnnagatc 60

ggaagagcac acgtctgaac tccagtca 88

<210> 8

<211> 89

<212> DNA

<213> Artificial sequence

<220>

<223> media barcode, oligo 3

<220>

<221> other_characteristic

<222> (1)...(5)

<223> phosphorothioate bond

<220>

<221> other_characteristic

<222> (85)...(89)

<223> phosphorothioate bond

<220>

<221> other_characteristic

<222> 41, 43, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 56

<223> n = A, T, C or G

<400> 8

tacgtctacg cgctgctctg ccacgactga cggacgtatt nwnnnnwnnn nwnnnnagat 60

cggaagagca cacgtctgaa ctccagtca 89

<210> 9

<211> 90

<212> DNA

<213> Artificial sequence

<220>

<223> media barcode, oligo 4

<220>

<221> other_characteristic

<222> (1)...(5)

<223> Phosphorothioate

<220>

<221> other_characteristic

<222> (86)...(90)

<223> Phosphorothioate

<220>

<221> other_characteristic

<222> 41, 42, 44, 45, 46, 47, 49, 50, 51, 52, 54, 55, 56, 57

<223> n = A, T, C or G

<400> 9

tacgtctacg cgctgctctg ccacgactga cggacgtatt nnwnnnnwnn nnwnnnnaga 60

tcggaagagc acacgtctga actccagtca 90

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> direct primer with carrier barcode

<400> 10

gtgactggag ttcagacgtg tgct 24

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer with carrier barcode

<400> 11

tacgtctacg cgctgctctg 20

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> RT IgG constant region primer

<220>

<221> modified base

<222> 10

<223>gm

<220>

<221> modified base

<222> 11

<223>gm

<220>

<221> modified base

<222> 12

<223>gm

<400> 12

aggacagccgggaaggtgt 19

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> IgL constant RT primer

<220>

<221> modified base

<222> 9

<223>gm

<220>

<221> modified base

<222> 11

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 12

<223>gm

<400> 13

gctcccgggt agaagtca 18

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> IgK constant RT primer

<220>

<221> modified base

<222> 11

<223>gm

<220>

<221> modified base

<222> 12

<223>gm

<220>

<221> modified base

<222> 13

<223> 2'-O-methyladenosine

<400> 14

ggcctctctg ggatagaagt 20

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> RT IgM constant region primer

<220>

<221> modified base

<222> 13

<223>gm

<220>

<221> modified base

<222> 14

<223>cm

<220>

<221> modified base

<222> 15

<223>gm

<400> 15

tgtgaggtgg ctgcgtactt g 21

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> RT IgA constant region primer

<220>

<221> modified base

<222> 12

<223>gm

<220>

<221> modified base

<222> 13

<223>gm

<220>

<221> modified base

<222> 14

<223> 2'-O-methyladenosine

<400> 16

ctggctrggt gggaagtttc t 21

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> RT IgD constant region primer

<220>

<221> modified base

<222> 12

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 13

<223> gmcm

<220>

<221> modified base

<222> 15

<223>cm

<400> 17

cacgcatttg tactcgcctt g 21

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> RT IgE constant region primer

<220>

<221> modified base

<222> 10

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 12

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 13

<223>gm

<400> 18

gatggtggca tagtgaccag 20

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> RT constant region primer TRA

<220>

<221> modified base

<222> 15

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 16

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 18

<223>cm

<400> 19

tgtttgagaa tcaaaatcgg tgaa 24

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> RT constant region primer TRB

<220>

<221> modified base

<222> 9

<223>gm

<220>

<221> modified base

<222> 10

<223>gm

<220>

<221> modified base

<222> 11

<223>gm

<400> 20

acgtggtcgg ggaagaag 18

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> RT TRG constant region primer

<220>

<221> modified base

<222> 13

<223>ac4cgm

<220>

<221> modified base

<222> 14

<223>gm

<220>

<221> modified base

<222> 16

<223> 2'-O-methyladenosine

<400> 21

caagaagaca aaggtatgtt cc 22

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> RT constant region primer TRD

<220>

<221> modified base

<222> 12

<223>gm

<220>

<221> modified base

<222> 13

<223> 2'-O-methyladenosine

<220>

<221> modified base

<222> 14

<223>cm

<400> 22

tcttcttgga tgacacgaga 20

<210> 23

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> matrix switching, oligo

<220>

<221> modified base

<222> 24

<223> 2'-deoxyuridine

<220>

<221> other_characteristic

<222> 34

<223> riboguanosine

<220>

<221> other_characteristic

<222> 35

<223> riboguanosine

<220>

<221> modified base

<222> 36

<223> 3-deoxyguanosine

<220>

<221> other_characteristic

<222> 25, 26, 28, 29, 31, 32

<223> n = A, T, C or G

<400> 23

aatacgtccg tcagtcgtgg atgunntnna nntggg 36

<210> 24

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> degenerate protrusion

<220>

<221> other_characteristic

<222> 1, 2, 3, 4, 5, 6

<223> n = A, T, C or G

<400> 24

nnnnnn 6

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequence "short P5"

<220>

<221> modified base

<222> 1

<223> phosphorylated adenosine

<220>

<221> modified base

<222> 20

<223> 3' amino-modified (3AmMO) thymine

<400> 25

agatcggaag agcgtcgtgt 20

<210> 26

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> hinged (splint) oligonucleotide

<220>

<221> modified base

<222> 26

<223> 3'amino modified (3AmMO) nucleotide (a, g, c or t)

<220>

<221> other_characteristic

<222> 21, 22, 23, 24, 25, 26

<223> n = A, T, C or G

<400> 26

acacgacgct cttccgatct nnnnnn 26

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> "short P5" (short P5) reverse primer

<400> 27

acacgacgct cttccgatct 20

<210> 28

<211> 64

<212> DNA

<213> Artificial sequence

<220>

<223> C7-index-P7 direct primer

<220>

<221> other_characteristic

<222> 25, 26, 27, 28, 29, 30

<223> n = A, T, C or G

<400> 28

caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 29

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IgG constant region reverse primer sequence

<400> 29

aagtagtcct tgaccaggca gc 22

<210> 30

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> IgL constant region reverse primer sequence

<400> 30

ggcttgaagc tcctcagagg a 21

<210> 31

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IgK constant region reverse primer sequence

<400> 31

aggcacacaa cagaggcagt tc 22

<210> 32

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IgM constant region reverse primer sequence

<400> 32

cgacggggaa ttctcacagg ag 22

<210> 33

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IgD constant region reverse primer sequence

<400> 33

tgtctgcacc ctgatatgat gg 22

<210> 34

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> IgA constant region 1 reverse primer sequence

<400> 34

gggtgctgca gaggctcag 19

<210> 35

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> IgA constant region 2 reverse primer sequence

<400> 35

gggtgctgtc gaggctcag 19

<210> 36

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IgE constant region reverse primer sequence

<400> 36

ggaatgtttt tgcagcagcg gg 22

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> TRA constant region reverse primer sequence

<400> 37

agtctctcag ctggtacacg g 21

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> TRB constant region reverse primer sequence

<400> 38

atggctcaaa cacagcgacc tc 22

<210> 39

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> C7

<400> 39

caagcagaag acggcatacg agat 24

<210> 40

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> IgG target-specific reverse primer sequence

with labeled adapter

<400> 40

acacgacgct cttccgatct ccagggggaa gacsgatg 38

<210> 41

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> IgG target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 41

acacgacgct cttccgatct nccaggggga agacsgatg 39

<210> 42

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> IgG target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 42

acacgacgct cttccgatct nnccaggggg aagacsgatg 40

<210> 43

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> IgG target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 43

acacgacgct cttccgatct nnnccagggg gaagacsgat g 41

<210> 44

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> IgA adapter-tagged target-specific reverse primer

sequence

<400> 44

acacgacgct cttccgatct ggctcagcgg gaagaccttg 40

<210> 45

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> IgA target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 45

acacgacgct cttccgatct nggctcagcg ggaagacctt g 41

<210> 46

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> IgA target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 46

acacgacgct cttccgatct nnggctcagc gggaagacct tg 42

<210> 47

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> IgA target-specific reverse primer sequence

with marked adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 47

acacgacgct cttccgatct nnnggctcag cgggaagacc ttg 43

<210> 48

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> IgE target-specific reverse primer sequence

with labeled adapter

<400> 48

acacgacgct cttccgatct gggaagacgg atgggctctg 40

<210> 49

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> IgE target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 49

acacgacgct cttccgatct ngggaagacg gatgggctct g 41

<210> 50

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> IgE target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 50

acacgacgct cttccgatct nngggaagac ggatgggctc tg 42

<210> 51

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> IgE target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 51

acacgacgct cttccgatct nnngggaaga cggatgggct ctg 43

<210> 52

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> IgM target-specific reverse primer sequence

with labeled adapter

<400> 52

acacgacgct cttccgatct gagacgaggt ggaaaagggt tg 42

<210> 53

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> IgM target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 53

acacgacgct cttccgatct ngagacgagg tggaaaaggg ttg 43

<210> 54

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> IgM target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 54

acacgacgct cttccgatct nngagacgag gtggaaaagg gttg 44

<210> 55

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> IgM target-specific reverse primer sequence

with marked adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 55

acacgacgct cttccgatct nnngagacga ggtggaaaag ggttg 45

<210> 56

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> IgD target-specific reverse primer sequence

with marked adapter

<400> 56

acacgacgct cttccgatct ggaacacatc cggagccttg 40

<210> 57

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> IgD target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 57

acacgacgct cttccgatct nggaacacat ccggagcctt g 41

<210> 58

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> IgD target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 58

acacgacgct cttccgatct nnggaacaca tccggagcct tg 42

<210> 59

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> IgD target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 59

acacgacgct cttccgatct nnnggaacac atccggagcc ttg 43

<210> 60

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> IgL target-specific reverse primer sequence

with labeled adapter

<400> 60

acacgacgct cttccgatct agggygggaa cagagtgac 39

<210> 61

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> IgL target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 61

acacgacgct cttccgatct nagggyggga acagagtgac 40

<210> 62

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> IgL target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 62

acacgacgct cttccgatct nnagggyggg aacagagtga c 41

<210> 63

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> IgL target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 63

acacgacgct cttccgatct nnnagggygg gaacagagtg ac 42

<210> 64

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> IgK target-specific reverse primer sequence

with labeled adapter

<400> 64

acacgacgct cttccgatct gacagatggt gcagccacag 40

<210> 65

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> IgK target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 65

acacgacgct cttccgatct ngacagatgg tgcagccaca g 41

<210> 66

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> IgK target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 66

acacgacgct cttccgatct nngacagatg gtgcagccac ag 42

<210> 67

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> IgK target-specific reverse primer sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 67

acacgacgct cttccgatct nnngacagat ggtgcagcca cag 43

<210> 68

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRA sequence

with labeled adapter

<400> 68

acacgacgct cttccgatct cacggcaggg tcagggttc 39

<210> 69

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRA sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 69

acacgacgct cttccgatct ncacggcagg gtcaggggttc 40

<210> 70

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRA sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 70

acacgacgct cttccgatct nncacggcag ggtcaggggtt c 41

<210> 71

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRA sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 71

acacgacgct cttccgatct nnncacggca gggtcagggt tc 42

<210> 72

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRB sequence

with labeled adapter

<400> 72

acacgacgct cttccgatct cgacctcggg tgggaacac 39

<210> 73

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRB sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21

<223> n = A, T, C or G

<400> 73

acacgacgct cttccgatct ncgacctcgg gtgggaacac 40

<210> 74

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRB sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22

<223> n = A, T, C or G

<400> 74

acacgacgct cttccgatct nncgacctcg ggtgggaaca c 41

<210> 75

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Target-specific reverse primer TRB sequence

with labeled adapter

<220>

<221> other_characteristic

<222> 21, 22, 23

<223> n = A, T, C or G

<400> 75

acacgacgct cttccgatct nnncgacctc gggtgggaac ac 42

<210> 76

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> C5-P5 reverse primer

<400> 76

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tcc 53

<210> 77

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> P7 priming site (C7)

<400> 77

agatcggaag agcacacgtc tgaactcca 29

<210> 78

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> P5 priming site

<400> 78

agatcggaag agcgtcgtgt agggaaagag tgtagatctc ggtggtcgcc gtatcatt 58

<210> 79

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Universal adapter

<220>

<221> other_characteristic

<222> 21, 22, 23, 24, 25, 26

<223> n = A, T, C or G

<400> 79

acacgacgct cttccgatct nnnnnn 26

<210> 80

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Universal adapter

<220>

<221> other_characteristic

<222> 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 13, 14

<223> n = A, T, C or G

<400> 80

nnnnwnnnnw nnnn 14

<210> 81

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Universal adapter

<220>

<221> other_characteristic

<222> 2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, 15

<223> n = A, T, C or G

<400> 81

wnnnnwnnnn wnnnn 15

<210> 82

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Universal adapter

<220>

<221> other_characteristic

<222> 1, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, 15

<223> n = A, T, C or G

<400> 82

nwnnnwnnnn wnnnn 15

<210> 83

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Universal adapter

<220>

<221> other_characteristic

<222> 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17

<223> n = A, T, C or G

<400> 83

nnwnnnnwnn nnwnnnn 17

<210> 84

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> IgM-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified thymine

<400> 84

tgtgaggtgg ctgcgtactt g 21

<210> 85

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> IgG-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified adenine

<400> 85

aggacagccgggaaggtgt 19

<210> 86

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> IgD-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified cytosine

<400> 86

cacgcatttg tactcgcctt g 21

<210> 87

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> IgA-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified cytosine

<400> 87

ctggctrggt gggaagtttc t 21

<210> 88

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> IgE-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified guanine

<400> 88

ggtggcatag tgaccagaga 20

<210> 89

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IgK-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified thymine

<400> 89

tattcagcag gcacacaaca ga 22

<210> 90

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> IgL-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified adenine

<400> 90

agtgtggcct tgttggcttg 20

<210> 91

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> TCR-A-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified guanine

<400> 91

gggagatctc tgcttctgat g 21

<210> 92

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> TCR-B-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified guanine

<400> 92

ggtgaatagg cagacagact tg 22

<210> 93

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> CD4-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified guanine

<400> 93

ggcagtcaat ccgaacact 19

<210> 94

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> CD-8-RT primer

<220>

<221> modified base

<222> 1

<223> biotin-modified cytosine

<400> 94

ctacaaagtg ggcccttctg 20

<210> 95

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> CD4 nested primer

<400> 95

acacgacgct cttccgatct tgtggccttg ccgagggagg 40

<210> 96

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> CD8 nested primer

<400> 96

acacgacgct cttccgatct tgcggaatcc cagagggcca 40

<210> 97

<211> 64

<212> DNA

<213> Artificial sequence

<220>

<223> C7-bc-P7 primer

<220>

<221> other_characteristic

<222> 25, 26, 27, 28, 29, 30

<223> n = A, T, C or G

<400> 97

caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 98

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> C5-P5 primer

<400> 98

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58

<210> 99

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Media barcode sequence

<220>

<221> other_characteristic

<222> 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 13, 14, 16, 17, 18, 19

<223> n = A, T, C or G

<400> 99

nnnnwnnnnw nnnnwnnnnw 20

<210> 100

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Media barcode sequence

<220>

<221> other_characteristic

<222> 1, 2, 3, 4, 6, 9, 10, 11, 13, 16, 17, 18

<223> n = A, T, C or G

<400> 100

nnnnwnscnn nwnscnnn 18

<---

Claims (179)

1. A method for obtaining a polynucleotide library, including: (a) lysing cells in each of a plurality of carriers, wherein each of said carriers comprises a cell belonging to a population of cells and further comprises a plurality of oligonucleotides carrying a molecular barcode, a single oligonucleotide or pool of oligonucleotides with a carrier barcode, and a first adapter or pool of first adapters; (b) producing in each carrier a plurality of complementary polynucleotide transcripts, said production comprising: (i) producing one or more target polynucleotides complementary to one or more target polynucleotides present in the cell using one or more target-specific primers; And (ii) creating a collection of polynucleotides, each of which is individually complementary to a polynucleotide transcript in the cell; (c) attaching to the plurality of complementary polynucleotides one of the plurality of oligonucleotides bearing a molecular barcode, thereby generating a plurality of molecular barcode polynucleotides each containing a molecular barcode; (d) attaching one of a single oligonucleotide or pool of oligonucleotides carrying a carrier barcode or amplified product thereof and a first adapter or one of a pool of first adapters or amplified product thereof to a plurality of molecular barcode polynucleotides, thereby generating a plurality of dual barcode polynucleotides; (e) obtaining a single-stranded amplicon from a plurality of double-barcoded polynucleotides; And (f) adding a second adapter to each of the single-stranded amplicons, wherein the first adapter and the second adapter are present at or near opposite ends of each of the double-barcoded single-stranded polynucleotides, where the first adapter and/or the second adapter contains at least one universal priming site. 2. The method of claim 1 wherein the collection of polynucleotides in (ii) is generated using random oligoprimers. 3. The method of claim 1 or 2, wherein the molecular barcode of each of the molecular barcoded polynucleotides is different from the molecular barcodes contained in the other molecular barcoded polynucleotides of the set. 4. The method according to any one of paragraphs. 1-3, wherein the molecular barcode of each of the molecular barcoded polynucleotides is a unique molecular barcode. 5. The method according to any one of paragraphs. 1-3, wherein in step (d) single-stranded dual-barcode polynucleotides each containing a molecular barcode and a carrier barcode are generated, each of the dual-barcoded polynucleotides of the same carrier containing the same carrier barcode. 6. The method according to any one of paragraphs. 1-5, including: (a) lysing cells in each of a plurality of carriers, wherein each of said carriers comprises a cell from a sample containing a population of cells, a plurality of molecular barcode oligonucleotides, a single oligonucleotide or pool of carrier barcoded oligonucleotides, and a first adapter or pool of first adapters; (b) producing in each carrier a plurality of complementary polynucleotide transcripts, said production of said plurality of complementary polynucleotides comprising (i) producing one or more target polynucleotides complementary to one or more target polynucleotide transcripts present in the cell; and (ii) creating a collection of polynucleotides, each of which is individually complementary to a polynucleotide transcript in the cell; (c) attaching to each complementary polynucleotide one of a plurality of oligonucleotides bearing a molecular barcode, thereby generating a plurality of barcoded polynucleotides each containing a unique molecular barcode; (d) attaching one of a single oligonucleotide or a pool of oligonucleotides carrying a carrier barcode or an amplified product thereof and a first adapter or one of a pool of first adapters or an amplified product thereof to each of the barcoded polynucleotides, thereby generating a plurality of double-barcoded polynucleotides, optionally single stranded dual-barcode polynucleotides, wherein each of the dual-barcode polynucleotides contains a molecular barcode and a carrier barcode, and wherein each of the dual-barcode polynucleotides belonging to the same carrier contains the same carrier barcode; (e) obtaining a single-stranded amplicon of each of the plurality of double-barcoded polynucleotides; And (f) adding a second adapter to each of the single-stranded amplicons, thereby adding a second adapter to the double-barcoded single-stranded polynucleotide, wherein the first adapter and the second adapter are present at or near opposite ends of each of the double-barcoded single-stranded polynucleotides. 7. The method according to any one of paragraphs. 1-6, where the first adapter includes a barcoded oligonucleotide. 8. The method according to any one of paragraphs. 1-7, wherein the collection of polynucleotides from each cell belonging to the cell population collectively includes sequences that are complementary to transcriptome or partial transcriptome transcripts of the cell. 9. The method of claim 8, wherein the transcriptome or partial transcriptome comprises at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the transcripts present in the cell genome. 10. The method according to any one of paragraphs. 1-9, in which one or more target polynucleotides from (i) and/or polynucleotides from (ii), on the basis of which the amplicon (i) and/or amplicon (II) are derived, is/derived from DNA. 11. The method according to any one of paragraphs. 1-10, in which one or more target polynucleotides from (i) and/or polynucleotides from (ii), on the basis of which the amplicon (i) and/or amplicon (II) are derived, is/derived from RNA. 12. The method of claim 11 wherein the RNA is mRNA. 13. The method according to any one of paragraphs. 1-12, in which each of the single or more complementary polynucleotides from (b) is cDNA. 14. The method according to any one of paragraphs. 1-13, wherein each of the single or more barcoded single stranded polynucleotides is a cDNA strand. 15. The method according to any one of paragraphs. 1-14, in which the first adapter and/or the second adapter contains at least one universal priming site. 16. The method according to any one of paragraphs. 1-15, in which: the first adapter and the second adapter are different; and/or the first adapter contains a first universal priming site, and the second adapter contains a second universal priming site, where optionally the first universal priming site and the second universal priming site are different. 17. The method of claim 16 wherein the first universal priming site and/or the second universal priming site is or contains a P7(C7) priming site or a continuous portion thereof, or a P5(C5) priming site or a continuous portion thereof, and wherein, optionally, a continuous portion of these is sufficient to anneal the complementary sequence. 18. The method of claim 16 or 17, wherein the first universal priming site is or contains a P7(C7) priming site or a contiguous portion thereof, and the second universal priming site is or contains a P5(C5) priming site or a continuous portion thereof . 19. The method of claim 17 or 18, wherein the P7(C7) priming site contains or is a contiguous portion of the sequence AGATCGGAAGAGCACACGTCTGAACTCCA (SEQ ID NO: 77). 20. The method according to claim 17 or 18, characterized in that the P5 priming site contains the sequence AGATCGGAAGAGCGTCGTGTAGGGAAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT (SEQ ID NO: 78) or is a contiguous part thereof. 21. The method according to any one of paragraphs. 17-20, wherein the contiguous portion is at least or at least about 15, 20, 25, or 30 nucleotides in length. 22. The method according to any one of paragraphs. 17, 18, 19 or 20, in which the P5 priming site is a contiguous portion as indicated in SEQ ID NO: 25 (AGATCGGAAGAGCGTCGTGT). 23. The method according to any one of paragraphs. 1-22, wherein the addition of a second adapter comprises hybridizing the splint oligonucleotide to each of the barcoded single-stranded polynucleotides in the presence of an oligonucleotide containing a second universal priming site, and wherein the splint oligonucleotide contains (i) a sequence complementary to the second universal priming site, and (ii) a degenerate overhang sequence capable of randomly annealing to the 3' end of the barcoded single stranded polynucleotide. 24. The method of claim 23, wherein prior to hybridization, the hinge oligonucleotide and the oligonucleotide containing the second universal priming site are annealed to form an adapter duplex with the hinge oligonucleotide. 25. The method of claim 23 or 24, wherein the degenerate overhang sequence comprises the sequence (N) _3-12 where N is any nucleotide. 26. The method according to any one of paragraphs. 23-25, in which the degenerate overhang sequence contains the sequence NNNNNN, where N is any nucleotide (SEQ ID NO: 24). 27. The method according to any one of paragraphs. 23-26, where the hinge (splint) oligonucleotide contains the sequence ACACGACGCTCTTCCGATCTNNNNNN, where N is any amino acid (SEQ ID NO: 26). 28. The method according to any one of paragraphs. 23-27, where the oligonucleotide containing the second universal priming site contains the sequence AGATCGGAAGAGCGTCGTGT (SEQ ID NO: 25). 29. The method according to any one of paragraphs. 1-28, wherein the carrier barcode oligonucleotide contains at least or at least about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 nucleotides. 30. The method according to any one of paragraphs. 1-29, wherein the carrier barcode oligonucleotide contains from or about 10 to 30 nucleotides. 31. The method according to any one of paragraphs. 1-30, wherein the carrier barcoded oligonucleotide contains a degenerate sequence. 32. The method according to any one of paragraphs. 1-31, wherein the carrier barcode oligonucleotide contains the sequence (N) _14-17 where N is any nucleotide and where optionally at least one or two Ns in the sequence are W, where W is adenine or thymine . 33. The method according to any one of paragraphs. 1-32, wherein the carrier barcode oligonucleotide contains the sequence NNNNWNNNNWNNNN (SEQ ID NO:80), WNNNNWNNNNWNNNN (SEQ ID NO:81), NWNNNWNNNNWNNNN (SEQ ID NO:82), or NNWNNNNWNNNNWNNNN (SEQ ID NO:83), where N is any nucleotide and W is adenine or thymine. 34. The method according to any one of paragraphs. 1-33, wherein each carrier comprises a pool of first adapters comprising a pool of carrier-barcoded oligonucleotides, wherein each carrier-barcoded oligonucleotide of the first adapter pool contains at least one base shift or added base, compared to at least one other of oligonucleotides with carrier barcode. 35. The method of claim 34, wherein the first adapter pool carrier-barcoded oligonucleotides contain the sequences NNNNWNNNNWNNNN (SEQ ID NO:80), WNNNNWNNNNWNNNN (SEQ ID NO:81), NWNNNWNNNNWNNNN (SEQ ID NO:82), and NNWNNNNWNNNNWNNNN (SEQ ID NO:82) :83), where N is any nucleotide and W is adenine or thymine. 36. The method according to any one of paragraphs. 1-35, wherein in step (d) the method further comprises amplifying: a single oligonucleotide or a pool of oligonucleotides with a carrier barcode; or a single adapter or a pool of first adapters, where the first adapters include a single oligonucleotide or a pool of oligonucleotides carrying a carrier barcode, where: the amplification is carried out before or simultaneously with the attachment of the carrier-barcoded oligonucleotide. 37. The method according to any one of paragraphs. 1-36, wherein the attachment of the carrier barcoded oligonucleotide comprises hybridizing a region of the carrier barcoded oligonucleotide with a region of each of the complementary polynucleotides or with a region of each of the molecular barcoded polynucleotides. 38. The method of claim 37, wherein such region comprises a 3' labeled/labeled polynucleotide complementary to the 3' end region of the molecular barcode of the polynucleotides bearing the molecular barcode. 39. The method according to any one of paragraphs. 1-38, wherein in step (b): one or more target polynucleotides are obtained by reverse transcription of the target polynucleotide(s) in the presence of reverse transcriptase and one or more target-specific primers complementary to the target sequence of the target polynucleotide(s); and/or a collection of polynucleotides is obtained by reverse transcription of the polynucleotide transcripts in the cell in the presence of reverse transcriptase and one or more transcriptome primers complementary to the polynucleotide transcript in the cell. 40. The method according to any one of paragraphs. 1-39, wherein the one or more target polynucleotides is a polynucleotide of an immune molecule or chain thereof. 41. The method according to any one of paragraphs. 1-40, wherein the one or more target polynucleotides comprise at least two target polynucleotides, each of which is an immune molecule chain polynucleotide. 42. The method according to any one of paragraphs. 1-41, wherein the one or more target polynucleotides comprise a TCR polynucleotide or chain thereof. 43. The method according to any one of paragraphs. 1-42, wherein the one or more target polynucleotides comprise a first polynucleotide, a T cell receptor alpha (TCRα) polynucleotide, and a second T cell receptor (TCRβ) polynucleotide. 44. The method according to any one of paragraphs. 1-42, wherein the one or more target polynucleotides comprise a first T cell receptor gamma (TCRγ) polynucleotide polynucleotide and a second polynucleotide, a T cell receptor delta (TCRdelta; TCR _δ ) polynucleotide. 45. The method according to any one of paragraphs. 1-41, wherein the one or more target polynucleotides comprise an antibody polynucleotide or chain thereof. 46. The method according to any one of paragraphs. 1-41 and 45, wherein the one or more target polynucleotides comprise a first immunoglobulin heavy chain (IgH) polynucleotide polynucleotide and a second immunoglobulin light chain (IgL) polynucleotide polynucleotide. 47. The method according to any one of paragraphs. 39-46, in which one or more target-specific primers and/or one or more transcriptome primers contain a poly(T) sequence. 48. The method according to any one of paragraphs. 39-46, wherein one or more transcriptome primers comprise a mixture of random hexamer oligonucleotide primers. 49. The method according to any one of paragraphs. 39-48, wherein the one or more target-specific primers comprise one or more primers that are complementary to the target sequence(s) of the target polynucleotide. 50. The method of claim 49, wherein the one or more target-specific primers include at least a first primer and a second primer. 51. The method of claim 49 or 50, wherein the one or more target-specific primers comprise at least primers to the target sequence of a plurality of target polynucleotides, each of which encodes an immune molecule or chain thereof. 52. The method of claim 51 wherein the immune molecule is a T cell receptor or an antibody. 53. The method according to any one of paragraphs. 50-52, in which at least the first primer is complementary to the target sequence of the polynucleotide of the first chain of the immune molecule, and the second primer is complementary to the target sequence of the polynucleotide of the second chain of the immune molecule. 54. The method according to any one of paragraphs. 50-53, wherein the first and second primers are complementary to the target sequences of different TCR chains of TCR polynucleotides. 55. The method according to any one of paragraphs. 50-54, in which: the first primer is complementary to the target sequence of the TCRalpha polynucleotide sequence and the second primer is complementary to the target sequence of the TCRbeta polynucleotide sequence; or the first primer is complementary to the target sequence of the TCRgamma polynucleotide sequence, and the second primer is complementary to the target sequence of the TCR-delta polynucleotide sequence. 56. The method of claim 54 or 55, wherein the target polynucleotide sequence of the TCR chains is a constant region sequence. 57. The method according to any one of paragraphs. 50-56, in which: the first primer is complementary to the target constant region of the TCR-alpha polynucleotide sequence, and the second primer is complementary to the target constant region of the TCR-beta polynucleotide sequence; or the first primer is complementary to the target constant region of the TCRgamma polynucleotide sequence, and the second primer is complementary to the target constant region of the TCR-delta polynucleotide sequence. 58. The method according to any one of paragraphs. 50-53, in which at least the first and second primers are complementary to target sequences of polynucleotides of different chains belonging to the antibody. 59. The method according to any one of paragraphs. 50-53 and 58, wherein the first primer is complementary to the target immunoglobulin heavy chain (IgH) polynucleotide sequence and the second primer is complementary to the target immunoglobulin light chain (IgL) polynucleotide sequence. 60. The method of claim 58 or 59, wherein the target polynucleotide sequence of the antibody chain sequence is a constant region sequence. 61. The method according to any one of paragraphs. 50-53 and 58-60, wherein the first primer is complementary to the constant region ( _CH ) target sequence of the heavy chain polynucleotide sequence and the second primer is complementary to the constant region ( _CL ) target sequence of the light chain polynucleotide sequence. 62. The method according to claim 60 or 61, in which the target polynucleotide _CH is derived from IgM, IgD, IgA, IgE or IgG, or combinations thereof; and/or the target sequence of the C _L polynucleotide sequence is from Igkappa, Iglambda, or combinations thereof. 63. The method according to any one of paragraphs. 1-62, in which one or more target polynucleotides contain a full-length coding sequence. 64. The method according to paragraphs. 1-63, in which one or more target polynucleotides and a collection of polynucleotide(s) are produced in the carrier in the same reaction volume. 65. The method according to any one of paragraphs. 1-64, in which in step (b) the production of a plurality of complementary polynucleotides includes the use of a non-template terminal transferase, and wherein three or more non-template nucleotides, ribonucleotides, or analogs thereof are added to the 3' end of each produced complementary polynucleotide. 66. The method of claim 65 wherein the non-template terminal transferase is a reverse transcriptase or polymerase. 67. The method of claim 65 or 66, wherein the non-template terminal transferase is a reverse transcriptase, and wherein the reverse transcriptase is selected from the list: Superscript II reverse transcriptase, Maxima reverse transcriptase, Protoscript II reverse transcriptase, Maloney mouse leukemia virus (MMLV) reverse transcriptase -RT), HighScriber reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, any reverse transcriptase having terminal deoxynucleotidyl transferase activity, and combinations thereof. 68. The method according to any one of paragraphs. 1-67, wherein in step (c) the addition comprises hybridizing a region of one of the plurality of oligonucleotides bearing the molecular barcode to three or more non-template nucleotides of each of the complementary polynucleotides. 69. The method of claim 68, wherein the plurality of oligonucleotides carrying the molecular barcode are provided as a plurality of template-switched oligonucleotides, each containing a 3' portion complementary to three or more non-template nucleotides. 70. The method of claim 69, wherein the template switch oligonucleotide also comprises a 5' end region complementary to a portion of the first adapter, the first adapter comprising the carrier barcoded oligonucleotide. 71. The method according to any one of paragraphs. 39-70, in which: reverse transcriptase has template switching activity; at least some of the many complementary polynucleotide chains produced contain a 3' overhang containing three or more non-template nucleotides; a plurality of oligonucleotides carrying a molecular barcode are presented as a plurality of template-switched oligonucleotides, each containing (1) a 5'-terminal region complementary to a 3'-labeled oligonucleotide containing a first adapter and an oligonucleotide with a carrier barcode, (2) a molecular barcode and (3) a 3'-part complementary to three or more non-template nucleotides of the 3'-overhang; And the template switch oligonucleotide serves as a template for reverse transcriptase so that a molecular barcode is included in each complementary polynucleotide to produce molecular barcode polynucleotides. 72. The method according to any one of paragraphs. 69-71, in which the 3'-part, complementary to three or more non-template nucleotides, contains a nucleotide, a ribonucleotide, or an analogue thereof. 73. The method according to any one of paragraphs. 65-72, in which three or more non-template nucleotides contain three or more C-nucleotides, and the 3'-part, complementary to three or more non-template nucleotides, contains one or more G-nucleotides or a ribonucleotide or an analogue thereof. 74. The method according to any one of paragraphs. 65-73, wherein the template-switch oligonucleotide further comprises a 3'-modified nucleotide that blocks extension of the template-switch oligonucleotide by reverse transcriptase or DNA polymerase. 75. The method of claim 74, wherein the modification is a deoxy, phosphate, amino, or alkyl modification of the 3'-terminal nucleotide. 76. The method according to any one of paragraphs. 1-75, wherein step (d) further comprises extending each of the plurality of molecular barcode polynucleotides after addition to generate the plurality of double barcode polynucleotides. 77. The method according to any one of paragraphs. 1-76, wherein the carrier is a well, emulsion, drop, or microcapsule. 78. The method according to any one of paragraphs. 1-77, comprising combining the contents of two or more of the plurality of carriers before step (e), resulting in a homogeneous mixture containing two or more of the plurality of double-barcoded single-stranded polynucleotides. 79. The method of claim 78, wherein combining the contents of the multiple carriers comprises disrupting two or more of the plurality of carriers and combining double-barcoded single-stranded polynucleotides from the two or more disrupted carriers. 80. The method of claim 78 or 79, comprising, prior to step (e), selecting or purifying single-stranded double barcode polynucleotides having a size greater than or greater than about 50 base pairs, greater than 100 base pairs, or greater than 200 base pairs. 81. The method according to any one of paragraphs. 78-80, comprising prior to step (e) selecting or purifying double-barcoded single-stranded polynucleotides having a size from or about 50 base pairs (bp) to 1500 bp, from 50 bp up to 1250 b.p., from 50 b.p. up to 1000 b.p., from 50 b.p. up to 750 b.p., from 50 b.p. up to 500 b.p., from 100 b.p. up to 1500 b.p., from 100 b.p. up to 1250 b.p., from 100 b.p. up to 1000 b.p., from 100 b.p. up to 750 b.p., from 100 b.p. up to 500 n.p., 200 n.p. up to 1500 b.p., from 200 b.p. up to 1250 b.p., from 200 b.p. up to 1000 b.p., from 200 b.p. up to 750 b.p. or from 250 n.p. up to 500 b.p. 82. The method according to any one of paragraphs. 1-81, wherein the double-barcoded single-stranded polynucleotides are in order (5' to 3'): first adapter, carrier barcode, molecular barcode, and second adapter. 83. The method according to any one of paragraphs. 1-82, wherein the first adapter is located at or near the 5' region of a single-stranded barcoded polynucleotide, optionally a double-barcoded single-stranded polynucleotide. 84. The method according to any one of paragraphs. 1-83, wherein the second adapter is located at or near the 3' region of a single-stranded barcoded polynucleotide, optionally a double-barcoded single-stranded polynucleotide. 85. The method according to any one of paragraphs. 21-84, wherein one or more steps (a)-(f) are carried out in solution and/or not carried out in the presence of a solid support, where optionally the solid support is or contains a bead. 86. The method according to any one of paragraphs. 19-85, wherein at least steps (c) and (d) are carried out in solution and/or not carried out in the presence of a solid support, where optionally the solid support is or contains a bead. 87. The method according to any one of paragraphs. 20-86, wherein each of steps (a)-(e) is carried out in solution and/or not carried out in the presence of a solid support, where optionally the solid support is or contains a bead. 88. The method according to any one of paragraphs. 1-87, wherein the cell population includes at least or at least about 1x10 ³ , 5x10 ³ , 1x10 ^{4 , 5x10 4} , 1x10 ⁵ , 5x10 ⁵ , 1x10 ^{6 ,} or 5x10 ⁶ cells. 89. The method according to any one of paragraphs. 1-88, wherein the cell population is derived from a biological sample obtained from a subject. 90. The method of claim 89, wherein the biological sample is or contains a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product . 91. The method according to any one of paragraphs. 1-90, where the cell population contains immune cells. 92. The method of claim 91 wherein the immune cells include lymphocytes or antigen presenting cells. 93. The method of claim 91 or 92, wherein the immune cell is a lymphocyte or subtype thereof, a B cell or subtype thereof, a T cell or subtype thereof, or a combination thereof. 94. The method of claim 93 wherein the immune cell is a T cell that is a CD4+ and/or CD8+ T cell. 95. The method according to any one of paragraphs. 1-94, wherein the cell population is enriched in or includes central memory T cells, effector memory T cells, naive T cells, stem central memory T cells, effector T cells, and regulatory T cells. 96. The method according to any one of paragraphs. 1-93, in which the cell population is enriched in memory B cells, naive B cells, or plasmablast B cells. 97. The method according to any one of paragraphs. 89-96, in which the subject is a human. 98. The method according to any one of paragraphs. 89-97, wherein the subject has cancer, infection, or an autoimmune condition. 99. The method of claim 98, wherein the infection is a viral, bacterial, or fungal infection. 100. The method according to any one of paragraphs. 1-99 further comprising amplifying a plurality of barcoded single strand polynucleotides, thereby generating a plurality of polynucleotide templates. 101. The method of claim 100 wherein amplification of the plurality of barcoded single strand polynucleotides is carried out in the presence of a first primer set comprising a first primer complementary to the first adapter sequence and a second primer complementary to the second adapter sequence. 102. The method of claim 101, wherein the first and/or second primer is a universal primer. 103. The method of claim 102, wherein the first and/or second primer is complementary to a P7(C7) priming site, or a contiguous portion thereof, or a P5(C5) priming site, or a continuous portion thereof. 104. The method of claim 102 or 103, wherein the first primer is complementary to the P7(C7) primer site or contiguous portion thereof and the second primer is complementary to the P5(C5) primer site or contiguous portion thereof. 105. The method according to item 103 or 104, in which the primer complementary to the P7(C7) priming site, or a contiguous portion thereof, has or includes the sequence CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO:39); and/or the primer complementary to the P5(C5) priming site, or a contiguous portion thereof, includes the sequence ACACGACGCTCTTCCGATCT (SEQ ID NO:27). 106. The method according to any one of paragraphs. 101-105, wherein the first and/or second primer further comprises a sequencing adapter. 107. The method of claim 106, wherein the primer complementary to the P7(C7) priming site or its contiguous portion further comprises the sequence CAAGCAGAAGACGGCATACGAGAT[NNNNNN]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:28); and/or the primer complementary to the P5(C5) primer site or its contiguous portion contains the sequence AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC (SEQ ID NO:76). 108. The method according to any one of paragraphs. 1-107, further comprising purifying each of the single-stranded barcoded polynucleotides, optionally double-barcoded single-stranded polynucleotides. 109. Polynucleotide library containing many barcoded polynucleotides obtained in accordance with the method according to any one of paragraphs. 1-108 for sequencing one or more of a plurality of target polynucleotides. 110. A method for sequencing one or more target polynucleotides and/or a complete or partial transcriptome of one or more cells, including sequencing one or more sets of double-barcoded polynucleotides obtained in accordance with the method according to any one of paragraphs. 1-108 or from a polynucleotide library according to item 109. 111. The method of claim 110, wherein the complete or partial transcriptome of one or more cells is sequenced. 112. The method of claim 111, further comprising amplifying the entire transcriptome or a portion thereof prior to sequencing. 113. The method of claim 112, wherein the amplification is performed using a first primer set containing a first primer and a second primer specific to the first and second adapter sequences, respectively. 114. The method according to any one of paragraphs. 110-113, in which one or more target polynucleotides are sequenced from a plurality of barcoded polynucleotides. 115. The method of claim 114, further comprising amplifying one or more target polynucleotides from the plurality of barcoded polynucleotides prior to sequencing. 116. The method of claim 115, wherein the full length sequence of one or more target polynucleotides is amplified. 117. The method of claim 115 or 116, wherein the amplification is carried out in the presence of a second primer set containing one or more first primers complementary to one or more target polynucleotides and a second primer complementary to the first adapter sequence. 118. The method of claim 117, wherein the second primer of the second primer set is complementary to a P7 (C7) priming site or a contiguous portion thereof or a P5 (C5) priming site or a continuous portion thereof. 119. The method of claim 117 or 118, wherein the second primer of the second primer set is complementary to the P7(C7) priming site or a contiguous portion thereof. 120. The method according to any one of paragraphs. 117-119, wherein the second primer of the second primer set represents or contains the sequence CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 39) or CAAGCAGAAGACGGCATACGAGAT[NNNNNN]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:28). 121. The method according to any one of paragraphs. 117-120, in which one or more first primers, complementary to one or more polynucleotides, are specific for the target sequence of the immune molecule or its chain. 122. The method of claim 121, wherein the immune molecule is a T cell receptor or an antibody. 123. The method of claim 121 or 122, wherein the one or more first primers are specific for the target sequence of the constant region of the immune molecule. 124. The method according to any one of paragraphs. 121-123, in which the immune molecule is a TCR and one or more first primers include AGTCTCTCAGCTGGTACACGG (SEQ ID NO: 37), ATGGCTCAAACACAGCGACCTC (SEQ ID NO: 38), or a combination thereof. 125. The method according to any one of paragraphs. 121-124, in which the immune molecule is an antibody, and where one or more of the first primers contain any of SEQ ID NO: 29-36 or a combination thereof. 126. The method according to any one of paragraphs. 110-125, further comprising determining the cellular origin of one or more barcoded polynucleotides, optionally double barcoded polynucleotides. 127. The method of claim 126, wherein determining the cell of origin comprises identifying double-barcoded polynucleotide sequences having the same carrier barcode indicating origin from the same cell. 128. The method according to p. 126 or 127, where the target polynucleotide is an immune molecule containing the first polynucleotide chain and the second polynucleotide chain, and the method includes matching the first polynucleotide chain and the second polynucleotide chain to the same cell based on the presence of the same the carrier barcode itself in the double-barcoded sequenced polynucleotides. 129. The method according to any one of paragraphs. 120-128, which further comprises quantifying or determining the number of polynucleotides with the same barcode, optionally with the same molecular barcode. 130. The method according to any one of paragraphs. 120-129, wherein the plurality of barcoded polynucleotides are double-barcoded polynucleotides containing a molecular barcode and a carrier barcode, and the method further comprises identifying transcriptome sequences and target polynucleotide sequences having the same carrier barcode, thereby providing information about the transcriptome of a carrier cell. target polynucleotide(s). 131. A method for analyzing a transcriptome, including: (a) sequencing one or more target polynucleotides from many belonging to a plurality of barcoded polynucleotides obtained by the method according to any one of paragraphs. 1-108, or from a plurality of barcoded polynucleotides of the polynucleotide library of claim 109, wherein the barcoded polynucleotides are dual-barcoded polynucleotides containing a molecular barcode and a carrier barcode, thereby generating target polynucleotide sequence information from a plurality of cells; (b) sequencing the entire transcriptome or part thereof from a plurality of barcoded polynucleotides obtained by the method of any one of paragraphs. 1-108, or from a plurality of barcoded polynucleotides of the polynucleotide library of claim 109, wherein the barcoded polynucleotides are dual-barcoded polynucleotides containing a molecular barcode and a carrier barcode, thereby generating transcriptome data from a plurality of cells; And (c) identifying sequence information from (a) and from (b) having the same carrier barcode as originating from the same cell. 132. A method for analyzing the transcriptome of a selected individual cell, including: (a) amplification and sequencing of one or more target polynucleotides from a plurality of barcoded polynucleotides obtained by the method of any one of paragraphs. 1-108, or from many belonging to a plurality of barcoded polynucleotides from the polynucleotide library of claim 109, wherein the barcoded polynucleotides are dual-barcoded polynucleotides containing a molecular barcode and a carrier barcode, thereby generating sequence information for each of the target polynucleotides of at least one of the many cells; (b) identifying the carrier barcode(s) associated with one of the target polynucleotide(s) sequenced in (a), thereby identifying the selected individual cell bearing the target polynucleotide; (c) amplifying and sequencing a transcriptome, or a portion thereof, from a plurality of cell barcoded polynucleotides bearing the carrier barcode identified in (b), thereby generating transcriptome data from the selected cell expressing the target polypeptide. 133. The method of claim 132, wherein the transcriptome or portion thereof from the selected cell is amplified or sequenced using a primer specific for the carrier barcode identified in (b) and a primer specific for the sequence of the second barcoded polynucleotide adapter. 134. A method for analyzing a transcriptome, which includes matching information about the sequence of a transcriptome or a part thereof and at least one of the target polynucleotides originating from the same cell, while the sequence information is determined on a set of barcoded polynucleotides obtained by the method according to any one of paragraphs. 1-106, or on a plurality of barcoded polynucleotides from the polynucleotide library of claim 109, wherein the barcoded polynucleotides are double-barcoded polynucleotides containing a molecular barcode and a carrier barcode, or determined by the method of any one of claims. 120-130. 135. The method of claim 134, wherein sequences having the same carrier barcode are determined to originate from the same cell. 136. The method according to any one of paragraphs. 131-135, wherein the transcriptome data contains a parameter, characteristic, trait, or phenotype associated with cell function or activity. 137. The method of claim 136, wherein the transcriptome data is associated with cell activation, depletion, or proliferative activity.

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