Methods of preparing nucleic acid libraries, and compositions and kits for practicing those methods

Abstract

To provide methods of preparing nucleic acid libraries including immune cell receptor repertoire libraries.SOLUTION: Aspects of the methods include producing one or more libraries, including, e.g., expression libraries and / or immune cell receptor repertoire libraries, from double stranded complementary DNA (cDNA) generated through a template-switching reaction involving an RNA sample. In some aspects, the methods include preparing a library from a single cell and / or a library indexed at the single cell level. Compositions and kits for use in practicing the methods are also provided.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] The development of next-generation sequencing (NGS) technologies has enabled the rapid extraction of useful genomic and transcriptomic information from nucleic acid libraries generated. These technologies are revolutionizing biotechnology and biomedical research, as high-throughput NGS technologies (such as lllumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent (Proton / PGM sequencing) and SOLiD sequencing) allow nucleic acid molecules to be sequenced faster and more cheaply than previously used Sanger sequencing.

[0002] These powerful sequencing techniques place a special emphasis on library preparation. Well-prepared reverse-transcribed complementary DNA (cDNA) libraries can be analyzed using NGS techniques for a variety of purposes. For example, depending on the library preparation technique, transcriptomic data can be utilized to perform differential expression analysis to identify genes that are up- or down-regulated in certain situations.

[0003] cDNA libraries can also be used for T cell receptor (TCR) profiling by NGS, which regulates T cell selection, function, and activation and determines which antigen peptide-major histocompatibility complex (MHC) complexes the T cell will respond to.

[0004] An individual's TCR repertoire is highly diverse, as the TCR α and β chain genes are derived in each cell from somatic recombination of the variable, diversity and joining (V(D)J) segments of the TCR gene. Profiling this TCR diversity will advance our understanding of immune repertoire dynamics and advance our knowledge of the nature of immune responses and the pathology of immune disorders. These insights will advance a variety of therapies, including the rapidly evolving field of immuno-oncology. Summary of the Invention

[0005] Methods for preparing a nucleic acid library are provided. Embodiments of the methods include generating one or more libraries, including, for example, an expression library and / or an immune cell receptor repertoire library, from double-stranded complementary DNA (cDNA) generated through a template switching reaction with an RNA sample. In some embodiments, the methods include preparing libraries from single cells and / or preparing libraries indexed at the single cell level. Compositions and kits for use in carrying out the methods are also provided. [Brief description of the drawings]

[0006] [Figure 1] 1 shows a schematic diagram of a method for preparing two nucleic acid libraries from a single RNA sample according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 shows a schematic diagram of a method for preparing an expression library and an immune cell receptor repertoire library from a single RNA sample according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 shows a schematic diagram of generating a product double-stranded nucleic acid using a template switching reaction. [Figure 4] 1A-1C provide examples of the use of tagmentation in library preparation methods that can be adapted for use in the methods of the present disclosure. [Diagram 5]FIG. 1 shows an overall schematic diagram illustrating the overall process used to generate libraries for use in combined differential gene expression analysis and TCR profiling according to an embodiment of the present disclosure. [Figure 6] FIG. 1 shows an overall schematic diagram illustrating the overall process used to prepare libraries for TCR profiling from single T cells, as described in the relevant examples presented herein. [Figure 7] As per the example shown in FIG. 6, the distribution of indexing oligos and pools of samples in a 96 well plate are illustrated. [Figure 8] Shown are data testing the performance of the immune cell profiling workflow, showing the percentage of sequencing reads generated from either single Jurkat cells or single cell equivalents of Jurkat RNA that mapped to the TCR-α CRD3 or TCR-β CDR3 regions. [Figure 9] Further data testing the performance of the immune cell profiling workflow are presented, showing the percentage of sequencing reads that mapped to expected Jurkat clonotypes. [Figure 10] FIG. 1 shows an overall schematic of the single cell TCR profiling workflow tested in the multi-sample nanodispenser (MSND) format. [Figure 11] 1 shows the results of alignment analysis of sequencing reads derived from a single Jurkat cell that were mapped to the CDR3 region of TCR-α or TCR-β. [Figure 12A] 1 shows overall details of a single cell TCR profiling workflow tested in a multi-sample nanodispenser (MSND) format according to an embodiment of the present invention. [Figure 12B] 1 shows overall details of a single cell TCR profiling workflow tested in a multi-sample nanodispenser (MSND) format according to an embodiment of the present invention. [Figure 12C] 1 shows overall details of a single cell TCR profiling workflow tested in a multi-sample nanodispenser (MSND) format according to an embodiment of the present invention. [Figure 12D] 1 shows overall details of a single cell TCR profiling workflow tested in a multi-sample nanodispenser (MSND) format according to an embodiment of the present invention. [Figure 13] Shown are alpha and beta receptor read counts from immune cell receptor sequencing libraries generated using the protocol of Figures 12A-12D as described in the experimental section below. [Figure 14A] Figure 1 shows exemplary data from a split WTA library generated using the protocol of Figures 12A-12D as described in the experimental section below. Gene body coverage data is shown, where the X-axis is normalized coverage across the entire gene and the Y-axis is total exonic reads mapped. An example of a CCRF-CEM cell is shown in the figure. [Figure 14B] Figure 12 shows exemplary data from split WTA libraries generated using the protocol in Figures 12A-12D as described in the experimental section below. Principal component analysis is shown of reads mapped from 5'DE libraries from TALL, CCRF and treated (PMA) CCRF cells as shown in Figure 13 (arrows indicate separate groups). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] As used herein, the term "hybridization conditions" refers to conditions under which a primer or other polynucleotide will specifically hybridize to a region of a target nucleic acid with which it has some degree of complementarity. The term "specific hybridization" refers to a condition where a primer specifically hybridizes to a target nucleic acid. Whether a primer specifically hybridizes to a target nucleic acid depends on factors such as the degree of complementarity between the polymer and the target nucleic acid, and the temperature at which hybridization occurs (as determined by the melting temperature (TM) of the primer). The melting temperature refers to the temperature at which half of the primer-target nucleic acid duplex remains hybridized and half of the duplex dissociates into single strands. The Tm of a duplex may be determined experimentally or predicted using the formula Tm=81.5+16.6(log10[Na+])+0.41(G+C content)-(60 / N), where N is the chain length and [Na+] is less than 1M. Sambrook and Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor NY, Ch. 10). Other, more advanced models that depend on a variety of parameters may be used to predict the Tm of a primer / target duplex in response to a variety of hybridization conditions. Approaches for performing specific nucleic acid hybridization are described, for example, in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,part I,chapter 2,“Overview of principles of hybridization and the strategy of nucleic acids The results can be found in “acid probe assays,” Elsevier (1993).

[0008] The terms "complementary" and "complementarity," as used herein, refer to a nucleotide sequence that base pairs non-covalently to the entirety or a region of a target nucleic acid (e.g., a region of a product nucleic acid). In canonical Watson-Crick base pairing, in DNA, adenine (A) base pairs with thymine (T) and guanine (G) base pairs with cytosine (C). In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T and G is complementary to C. In RNA, A is complementary to U and U is complementary to A. Typically, "complementary" refers to a nucleotide sequence that is at least partially complementary. The term "complementary" also includes duplexes that are completely complementary, such that every nucleotide of one strand is complementary to every nucleotide of the other strand at the corresponding position. In certain cases, a nucleotide sequence may be partially complementary to a target, where not all nucleotides are complementary to all nucleotides of the target nucleic acid at all corresponding positions. For example, a primer may be completely (i.e., 100%) complementary to a target nucleic acid, or the primer and target nucleic acid may have a degree of complementarity that is not complete (e.g., 70%, 75%, 85%, 90%, 95%, 99%).

[0009] The percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison (e.g., gaps can be introduced into the sequence of the first sequence for optimal alignment). Nucleotides at corresponding positions are then compared, and the percent identity of the two sequences is derived from the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions / total number of positions×100). When a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, the molecules are identical at that position. A non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When using the BLAST and Gapped BLAST programs, Default parameters of the program (e.g., NBLAST) can be used. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be altered (e.g., wordlength=5 or wordlength=20).

[0010] A domain refers to a stretch or length of a nucleic acid that is composed of multiple nucleotides and that confers a distinct function to the nucleic acid. Examples of domains include barcoded unique molecular identifier (BUMI) domains, primer binding domains, hybridization domains, barcode domains (such as source barcode domains), unique molecular identifier (UMI) domains, next generation sequencing (NGS) adaptor domains, NGS indexing domains, and the like. In some cases, the terms "domain" and "region" may be used synonymously, for example, when describing a domain / region of an immune receptor chain (e.g., immune receptor constant domain / region, and the like). The length of a given domain may vary, but in some cases, the length ranges from 2 to 100 nt, such as 5 to 50 nt, for example, 5 to 30 nt.

[0011] Methods for preparing a nucleic acid library are provided. Embodiments of the methods include generating one or more libraries, including, for example, an expression library and / or an immune cell receptor repertoire library, from double-stranded complementary DNA (cDNA) generated through a template switching reaction with an RNA sample. In some embodiments, the methods include preparing libraries from single cells and / or preparing libraries indexed at the single cell level. Compositions and kits for use in carrying out the methods are also provided.

[0012] Before describing the disclosed method in more detail, it should be understood that the disclosed method is not limited to the specific embodiments described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the disclosed method will be limited only by the appended claims.

[0013] Where a range of values ​​is given, it is understood that, to the nearest tenth of the lower limit of the range, each value between the upper and lower limits of the range, and any other stated or intervening value in the stated range, is included in the methods of the disclosure, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller range, and are also included in the methods of the disclosure, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods of the disclosure.

[0014] In this specification, certain ranges are indicated by numerical values ​​followed by the term "about". The term "about" is used herein to literally encompass the exact number to which the term is attached, as well as numbers that are close to or approximately the value to which the term is attached. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately may be a number that, in the context in which the number is stated, will yield a substantially equivalent value to the specifically stated number.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed method belongs. Although any methods similar or equivalent to those described herein can be used in the practice or testing of the methods of the present disclosure, representative illustrative methods and materials are described below.

[0016] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein for the purpose of disclosing and describing the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods are not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0017] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to the exclusion of any optional element. As such, this statement is intended to serve as an underlying antecedent for use of exclusive terminology, such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

[0018] It is clear that certain features of the method of the present disclosure, which are described in the context of separate embodiments for clarity, may be combined in one embodiment. Conversely, various features of the method of the present disclosure, which are described in the context of one embodiment for brevity, may be combined separately or in any suitable subcombination. Any combination of embodiments is expressly included in the present invention and is disclosed herein to the extent that such combination includes an operable process and / or apparatus / system / kit, as if each combination were individually and expressly disclosed herein. In addition, all subcombinations listed in the embodiments that describe the variables thereof are also expressly included in the method of the present invention and are disclosed herein as if each such subcombination were individually and expressly disclosed herein.

[0019] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the inventive method. Any method shown can be carried out in the order of events shown or in any other order which is logically possible.

[0020] method As outlined above, the method for preparing one or more nucleic acid libraries is provided.One or more libraries can be prepared from a single RNA sample.For example, in some cases, one immune cell receptor repertoire library can be prepared from an RNA sample.In some cases, an expression library and an immune cell receptor repertoire library are prepared from a single RNA sample.

[0021] When the disclosed methods include preparing multiple libraries from a single sample, the multiple libraries may be prepared sequentially or simultaneously. For example, in some cases, the disclosed methods may include preparing an expression library and an immune cell receptor repertoire library simultaneously. "Preparing simultaneously" refers to preparing two or more libraries simultaneously. "Preparing sequentially" means that one or more library preparation steps (e.g., amplification, end capture, etc.) performed in preparing the expression library are performed simultaneously or at least partially overlap in time. In some cases, the disclosed method may include preparing the expression library and the immune cell receptor repertoire library sequentially (e.g., preparing the expression library before the immune cell receptor repertoire library, or preparing the immune cell receptor repertoire library before the expression library). "Preparing sequentially" means that the preparation of the two libraries does not overlap in time, e.g., one or more library preparation steps performed in preparing the two libraries are not performed simultaneously. In some cases, the sequential preparation of libraries may include completing the first library before starting the preparation of the next library. Thus, in some cases, the simultaneous preparation of libraries may include preparing the second library before the preparation of the first library is completed.

[0022] When multiple libraries are prepared from a single RNA sample, either sequentially or simultaneously, the disclosed method may include generating a product double-stranded cDNA from the RNA sample and then splitting the generated product double-stranded cDNA into a first reaction mixture and a second reaction mixture. For example, as outlined in FIG. 1, the RNA sample (100) may be used in a template switching reaction to generate double-stranded cDNA (101) from the RNA sample. The generated double-stranded cDNA (which may or may not be amplified, as desired) may then be split, for example, into two reactions and then used to generate a first library (102) and a second library (103), as shown. In some embodiments, multiple reaction mixtures containing the split double-stranded cDNA may be used to generate an expression library (200) and an immune cell receptor repertoire library (201), as shown in FIG. 2. Thus, if desired, such split reaction mixtures may be used separately to generate multiple libraries (including, for example, using a first reaction mixture to generate an expression library and a second reaction mixture to generate an immune cell receptor repertoire library).

[0023] The generated product double-stranded cDNA may be split evenly or unequally to provide equal or unequal amounts of the generated product double-stranded cDNA in the first and second reaction mixtures. For example, in some cases, the reaction mixture used to generate the expression library may contain more split product double-stranded cDNA than the reaction mixture used to generate the immune cell receptor repertoire library. In some cases, the reaction mixture used to generate the expression library may contain less split product double-stranded cDNA than the reaction mixture used to generate the immune cell receptor repertoire library. In some cases, the reaction mixture used to generate the expression library may contain the same amount of split product double-stranded cDNA as the reaction mixture used to generate the immune cell receptor repertoire library.

[0024] The product double-stranded cDNA may be split into two reaction mixtures as described above, but the splitting of the product double-stranded cDNA is not necessarily limited to splitting into two reaction mixtures, and in some cases, the product double-stranded cDNA may be split into more than two reaction mixtures. For example, in some cases, the product double-stranded cDNA may be split into three or more reaction mixtures. Splitting the product double-stranded cDNA into more than two reaction mixtures may be used for various purposes (for example, but not limited to, three or more libraries may be made from the product double-stranded cDNA (including product double-stranded cDNA made from a single RNA sample)).

[0025] The methods of the invention may use any convenient method of dividing the generated product double-stranded cDNA into different reaction mixtures. For example, in some cases, the product double-stranded cDNA may be The product double-stranded cDNA may be divided into reaction mixtures manually, for example, by manually pipetting an aliquot of the product double-stranded cDNA into the reaction mixture. In some cases, the product double-stranded cDNA may be divided into reaction mixtures automatically, for example, through the use of a liquid-handling robot or other automated device programmed to dispense an aliquot of the product double-stranded cDNA into the reaction mixture. In some cases, the methods of the disclosure may include pre-amplifying the product double-stranded cDNA, for example, through a pre-amplification PCR step, before dividing into reaction mixtures used to generate the individual libraries.

[0026] In some cases, the reaction mixtures prepared separately may be pooled before further processing, and thus the method may include a pooling step. For example, in some cases, the product double-stranded cDNAs prepared separately may be pooled before preparing one or more libraries. In some cases, the prepared product double-stranded cDNAs may be pooled before splitting the product double-stranded cDNA into separate reactions, for example, to prepare two or more libraries, as described above. Reactions prepared separately from separate single RNA samples (including, for example, a single RNA sample prepared from multiple cells, or a single RNA sample prepared from a single cell) may be pooled. Any convenient pooling method may be used (including, for example, pooling the entire volume of the reactions together, or pooling a partial volume of the reactions together). In some cases, the separate reactions may be performed in separate vessels or wells (e.g., wells of a multi-well plate), and the reaction mixtures of the wells of the vessels may be pooled into one vessel or well.

[0027] In some cases, nucleic acids produced in separate reactions and then pooled may contain or be modified to contain identifying nucleic acid sequences that allow for traceability back to the source of the separate nucleic acid reactions after pooling. Useful identifying nucleic acid sequences include, for example, barcode nucleic acid sequences and indexing sequences, as described in more detail below.

[0028] As described above, one or more libraries may be generated after the product double-stranded cDNA is generated from the RNA sample. The RNA sample is a sample that includes one or more types of template RNA, as described in more detail below. The RNA sample may be derived from a cell sample (including a single cell, or a cell sample that includes a cell population, for example, including two or more cells). The cell sample may be derived from a variety of sources, including, but not limited to, cell tissue, biopsy specimen, blood sample, cell culture fluid, and the like. In addition, the cell sample may be derived from a specific organ, tissue, tumor, neoplasm, and the like. Furthermore, cells from any population, such as a population of prokaryotic or eukaryotic single-cell organisms (including bacteria or yeast), can be the source of the cell sample used in the disclosed method. However, when the disclosed method includes preparing an immune cell receptor repertoire library, eukaryotic cells (including mammalian cells) will generally be used as the source of the RNA sample.

[0029] Thus, in some cases, the source of the RNA sample used in the methods of the present disclosure may be a mammalian cell sample, such as a rodent (e.g., mouse or rat) cell sample, a non-human primate cell sample, a human cell sample, etc. In some cases, the mammalian cell sample may be a mammalian blood sample, including, but not limited to, a rodent (e.g., mouse or rat) blood sample, a non-human primate blood sample, a human blood sample, etc.

[0030] In some embodiments, useful cell samples may include samples containing one or more immune cell types. As used herein, the term "immune cells" generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells), These include bone marrow derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of CD3 expressing immune cells, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Tregs) and gamma delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells and neutrophils, which are capable of mediating cytotoxic responses.

[0031] In some cases, the RNA sample used in the methods described herein will be derived from a population of immune cells, including but not limited to, a mixed population of immune cells, a population of T cells, a population of B cells, etc. In some cases, the RNA sample used in the methods described herein will be derived from a single immune cell, including but not limited to, a single T cell, a single B cell, etc.

[0032] Library As mentioned above, the library produced by the method of the present disclosure may be produced from the product double-stranded cDNA produced. "Product double-stranded cDNA" generally refers to double-stranded DNA produced from a reverse transcription reaction that contains the complement of a template nucleic acid. Product double-stranded cDNA may be produced from a template RNA using a reverse transcription reaction, in which case any RNA template may be used, including, for example, an mRNA template. Thus, the provided method may include producing product double-stranded cDNA from a template RNA present in an RNA sample through the use of a reverse transcription reaction (such as a template switching reverse transcription reaction, which is described in more detail below).

[0033] In some cases, the disclosed methods include preparing multiple libraries, such as multiple expression libraries, multiple immune cell receptor repertoire libraries, combinations thereof, etc., from multiple single cells. For example, in some cases, multiple individual RNA samples may each be derived from a single cell (e.g., including individual immune cells), and the individual RNA samples may be used to prepare product double-stranded cDNAs that are then used to generate multiple libraries. When multiple libraries are generated, the components used in the preparation of the libraries (e.g., product double-stranded cDNAs) or the libraries themselves may or may not be pooled. As described above and in more detail below, when libraries or library preparation components are pooled, the nucleic acids may include identifying non-template nucleic acid sequences that can be used to trace back to identify a particular library component or the source of that sequence. Such trace back identification may be performed, for example, through demultiplexing.

[0034] In some embodiments, the method aspect of the present disclosure comprises preparing an expression library. By "expression library" is meant a nucleic acid library that is useful for evaluating the nucleic acid expression of a cell sample (including, for example, a single cell sample, or a sample that comprises a population of cells). The preparation of an expression library can comprise preparing an expression library for next generation sequencing (NGS), including preparing an NGS expression library from an RNA sample.

[0035] An NGS library generated as described herein is a library whose nucleic acid members contain partial or complete sequencing platform adapter sequences at their termini that are useful for sequencing using a relevant sequencing platform, such as Illumina®'s HiSeq™, MiSeq™, and Genome Analyzer™ sequencing systems, Ion Torrent™'s Ion PGM™ and Ion Proton™ sequencing systems, and Pacific Biosciences' PACBIO RS II Sequel system. Sequencing systems that may be used include, but are not limited to, the NIH Sequencing System, Life Technologies' SOLiD Sequencing System, Roche's 454 GS FLX+ and GS Junior Sequencing Systems, Oxford Nanopore's MinION™ System, or any other applicable sequencing platform.

[0036] As described above, the disclosed method includes generating a product double-stranded cDNA from an RNA sample, which may be divided into two or more reaction mixtures, one of which may be used to prepare an expression library. The prepared expression library may be a full-length expression library or a non-full-length expression library. By "full-length expression library" it is meant that the nucleic acid members of the library contain a full-length cDNA sequence corresponding to the full-length RNA member that was reverse transcribed to generate the cDNA. For example, if an individual library member is a full-length cDNA of an mRNA, the full-length cDNA will contain the entire coding sequence of that mRNA, e.g., the entire spliced ​​mRNA coding sequence, i.e., the entire mRNA coding sequence between the 5' cap and the poly(A) tail of the mRNA. The full-length cDNA may or may not include sequences corresponding to one or more untranslated regions (UTRs) of the mRNA, e.g., the 3'UTR or 5'UTR.

[0037] The prepared expression library may in some cases be a library specifically prepared to capture the ends of the RNA molecules of interest. Such libraries may be referred to herein as "end-captured" libraries, or their members may be referred to as end-captured nucleic acids. End-captured libraries include nucleic acids that are separately subjected to 3'-end capture or 5'-end capture, and both 3'-end capture and 5'-end capture are performed on those nucleic acids. End-capture may utilize end amplification primers. As used herein, the term "end amplification primer" generally refers to a nucleic acid primer used in a PCR reaction to amplify from an end introduced into the double-stranded DNA to be amplified. The terminus introduced into the double-stranded DNA to which the terminal amplification primer binds will generally not be the original terminus of that double-stranded DNA (e.g., not the original 5' terminus (e.g., the original 5' terminus corresponding to the original 5' terminus of the reverse transcribed RNA) or not the original 3' terminus (e.g., the original 3' terminus corresponding to the original 3' terminus of the reverse transcribed RNA)) but may be a newly introduced terminus, e.g., an end created as the product of a fragmentation and / or ligation reaction.

[0038] Thus, in certain embodiments, the method for preparing an expression library is end capture. End capture may be used for sequencing and / or quantification of RNA (e.g., mRNA transcripts), such as differential expression analysis. End capture may utilize a tagmentation reaction, in which the double-stranded DNA of interest is fragmented and the fragments are ligated to a desired oligonucleotide containing a synthetic sequence (e.g., one or more of the non-template sequences described herein). Tagmentation may be performed through the use of a transposase that mediates this fragmentation and ligation.

[0039] In certain embodiments, the end capture method captures the 3' end of the RNA, e.g., end capture is facilitated by the presence of an amplification primer binding site in the first strand cDNA primer and a 5' post-tagmentation PCR primer binding site introduced by tagmentation. In other embodiments, the end capture method captures the 5' end of the RNA, e.g., end capture is facilitated by the presence of an amplification primer binding site in the template switch oligonucleotide and a 3' post-tagmentation PCR primer binding site introduced by tagmentation.

[0040] An example of a method for preparing an end-capture expression library is shown diagrammatically in Figure 4. The method includes combining an RNA sample, a first strand cDNA primer comprising a PCR primer binding domain, a template switch oligonucleotide comprising a 3' hybridization domain and a 5' second PCR primer binding domain, a reverse transcriptase (not shown), and dNTPs (not shown) in a reaction mixture under conditions sufficient to generate a double-stranded product nucleic acid (not shown) comprising a template mRNA and a template switch oligonucleotide hybridized to adjacent regions of the first strand cDNA. In this example, the RNA sample comprises an mRNA (polyA+) template, and the first strand cDNA primer comprises an oligo dT 3' hybridization domain, a barcode, and a sequencing adaptor domain (in this example, an Illumina® Read 4). The cDNA sequence includes a 3′ hybridization domain that includes an LNA and a 5′ domain that includes a second PCR primer binding domain (Primer2 sequence), a first PCR primer binding domain (in this example, a domain that binds Clontech® Primer IIA), and a blocking modification (black star). During first strand synthesis, the reverse transcriptase template switches from the template mRNA to a template switch oligonucleotide (in this example, a Clontech SMART-Seq v4 template switch oligonucleotide) that includes an LNA-containing 3′ hybridization domain and a 5′ domain (containing the second PCR primer binding domain). In this example, the second PCR primer binding domain (the domain that binds Clontech® Primer IIA) is the same as the first PCR primer binding domain. After first strand synthesis, the cDNA is amplified by PCR using the blocked Clontech® Primer IIA to generate the product double-stranded cDNA (labeled “double-stranded cDNA” in FIG. 4).

[0041] In the example shown in FIG. 4, the generation of product double-stranded cDNA is illustrated for ease of identification of primer binding domains and barcode sequences for downstream amplification and sequencing for reference. As described in more detail below, tagmentation used in the provided methods may differ from that shown in FIG. 4 in the presence and location of various elements (e.g., non-template sequences). For example, while the schematic diagram in FIG. 4 shows 3' end capture, the components of the various steps of the disclosed methods can be easily rearranged for 5' end capture. In addition, as described above, the provided methods may generally include generating product double-stranded cDNA and then splitting the generated double-stranded cDNA into a reaction mixture prior to tagmentation. Further description of library generation involving tagmentation reactions is provided in International Application No. PCT / US2016 / 051989, the disclosure of which is incorporated herein by reference in its entirety.

[0042] The expression library to be produced may exhibit a desired degree of complexity (e.g., high complexity). The "complexity" of an expression library refers to the proportion of redundant sequencing reads (e.g., reads sharing the same start site) obtained by sequencing the library. Complexity is inversely correlated with the proportion of redundant sequencing reads. In a low complexity library, certain target sequences are over-represented, while other targets (e.g., mRNAs expressed at low levels) suffer from low or no coverage. In a high complexity library, the sequencing reads closely approximate the known distribution of target nucleic acids in the starting nucleic acid sample, for example, providing coverage of targets (e.g., mRNAs expressed at low levels) known to be present at relatively low levels in the starting sample. According to certain embodiments, the complexity of the expression library generated according to the provided method is such that sequencing reads are generated for 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the target nucleic acids of different species (e.g., mRNAs of different species) in the starting nucleic acid sample (e.g., RNA sample). The complexity of the library may be determined by mapping the sequencing reads to a reference genome or transcriptome (e.g., a particular cell type). Daley et al. (201 3) Specific approaches have been developed to determine the complexity of sequencing libraries, including the approach described in Nature Methods 10(4):325-327.

[0043] In certain embodiments, the methods provided further comprise subjecting the prepared expression library to an NGS protocol, which may be performed on any suitable NGS sequencing platform, including sequencing platforms provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ sequencing systems), sequencing platforms provided by Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems), Pacific The sequencing platform may include, but is not limited to, sequencing platforms provided by Biosciences (e.g., PACBIO RS II Sequel Sequencing System), sequencing platforms provided by Life Technologies™ (e.g., SOLiD Sequencing System), sequencing platforms provided by Roche (e.g., 454 GS FLX+ and / or GS Junior Sequencing System), or any other suitable sequencing platform. NGS protocols will vary depending on the specific NGS sequencing system used. Detailed protocols for sequencing NGS libraries (which may include, for example, further amplification (e.g., solid-phase amplification), sequencing the amplicons, and analyzing the sequencing data) are available from the manufacturer of the NGS sequencing system used.

[0044] In certain embodiments, the methods of the present disclosure may be used to generate expression libraries corresponding to mRNAs for downstream sequencing on a relevant sequencing platform (e.g., sequencing platforms provided by Illumina®, Ion Torrent™, Pacific Biosciences, Life Technologies™, Roche, etc.). According to certain embodiments, the methods of the present disclosure may be used to generate NGS libraries corresponding to non-polyadenylated RNAs for downstream sequencing on a relevant sequencing platform. For example, as described elsewhere herein, microRNAs may be polyadenylated and then used as templates in template-switched polymerization reactions. Depending on the researcher's goals, random or gene-specific priming may be used. This library may be mixed 50:50 with a control library (e.g., Illumina® PhiX control library) and sequenced on a sequencing platform (e.g., Illumina® sequencing system). Control library sequences may be removed and the remaining sequences mapped to the transcriptome of the source of the mRNA (eg, human, mouse, or any other mRNA source).

[0045] The prepared expression libraries may be used in a variety of downstream analyses, and in some cases, the library preparation may be specifically reconfigured for the desired type of downstream analysis. For example, in some cases, the prepared expression libraries may be subjected to Whole Transcriptome Analysis (WTA), which includes analysis of mRNA and analysis of RNA species other than mRNA, such as non-coding RNA (e.g., snRNA and snoRNA). Thus, in some cases, the library preparation may include, for example, modification of poly(A) tails to allow analysis of RNA other than mRNA in the transcriptome. It may be custom constructed by using hybridization-independent primers (e.g., random primers) or by adding a tailing reaction, e.g., by adding a poly(A) tail to an RNA species that is not naturally polyadenylated prior to generation of the product double-stranded cDNA.

[0046] In some cases, preparation of a library, e.g., a library for WTA, may include a step of reducing the amount of ribosomal RNA in the sample and / or library. For selective removal, any convenient method of reducing and / or removing unwanted ribosomal RNA may be used, such as, for example, the use of affinity purification, degradation of contaminating nucleic acids (e.g., RiboGone™ (Takara Bio USA Inc., Mountain View, CA)). View, CA) and utilizing the methods described in U.S. Pat. No. 9,428,794 and U.S. Patent Application Publication No. 2015 / 0225773 A1, the disclosures of which are incorporated by reference in their entireties herein), and combinations thereof.

[0047] In certain embodiments, the prepared expression library may be used in differential expression analysis of one or more genes, including, for example, determining relative expression levels (i.e., up-regulation or down-regulation). Differential expression may be determined qualitatively or quantitatively, and such analysis may be transcriptome-wide or targeted. Thus, the number of expressed transcripts evaluated in the differential expression analysis of the present disclosure will vary. In some cases, the differential expression analysis may evaluate 50% or more of the expressed transcripts in a genome of interest (e.g., including but not limited to, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or essentially all of the expressed transcripts in a genome of interest). Targeted differential expression analysis may include analysis of only a subset or specific categories of transcripts. The transcript categories that may be limited to targeted expression analysis will vary, and may include, for example, but not limited to, immune gene transcripts.

[0048] Useful categories and subcategories of immune genes generally include genes responsible for immune system function and effective defense against pathogens, including, but not limited to, genes associated with immune system processes (e.g., genes identified by Gene Ontology (GO) accession number GO:0002376, available online at geneontology.org), such as, but not limited to, genes involved in B cell mediated immunity, B cell selection, T cell mediated immunity, T cell selection, activation of the immune response, antigen processing and presentation, antigen sampling in mucosa-associated lymphoid tissue, basophils, and the like. These include, but are not limited to, genes related to immune system activation, immune cell activation, immune cell homeostasis, immune cell mediated immunity, eosinophil mediated immunity, blood cell differentiation, blood cell proliferation, immune effector processes, immune response, immune system triggering, immune memory processes, leukocyte activation, leukocyte homeostasis, leukocyte mediated immunity, leukocyte migration, lymphocyte costimulation, lymphocyte mediated immunity, mast cell mediated immunity, myeloid cell homeostasis, myeloid leukocyte mediated immunity, natural killer cell mediated immunity, negative regulation of immune system processes, neutrophil mediated immunity, positive regulation of immune system processes, production of mediator molecules of immune response, regulation of immune system processes, diversification of immune receptors at the somatic cell level, tolerance induction, etc. Specific relevant genes include, but are not limited to, cytokines, interleukins, interleukin receptors, CD4, CD8, CD3, PD-1, etc.

[0049] As outlined above, in some embodiments, the disclosed methods include preparing an immune cell receptor repertoire library from an RNA sample. Aspects of the disclosed methods include amplifying immune cell receptor-specific cDNA from the product double-stranded cDNA generated from the RNA sample to generate an immune cell receptor repertoire library. An "immune cell receptor repertoire library" generally refers to a library of immune cell receptors that is generated from a type of cell or cell population. By "immunoreceptor repertoire library" is meant a nucleic acid library that comprises full-length or partial sequences of one or more immune receptors. For example, immune cell receptor repertoire library can be made in a single cell or in a cell population derived from a single cell sample, a single subject or a cell sample population (including, for example, a sample population derived from two or more subjects). In some cases, the library of the present disclosure can be made from individual single cells and pooled after adding identifying nucleic acid sequences.

[0050] As mentioned above, the members of the immune cell receptor repertoire library may vary in length and may be full-length or less than full-length. In some cases, the members of the library of the present disclosure will preferentially comprise the 5' end of an immune cell receptor. Examples of immune cell receptors of interest include, but are not limited to, T cell receptors (TCRs) and B cell receptors (BCRs).

[0051] In some cases, the immune cell receptor repertoire library may include a TCR repertoire library. The TCR complex is a disulfide-bonded membrane-anchored heterodimeric protein that is typically expressed on the surface of T cells and is composed of highly variable alpha (α) and beta (β) chains expressed as part of a complex with a CD3 chain molecule. Many native TCRs exist in the heterodimeric αβ or γδ forms. The complete endogenous TCR complex in the heterodimeric αβ form includes eight chains: an alpha chain (herein referred to as TCRα or TCR alpha), a beta chain (herein referred to as TCRβ or TCR beta), a delta chain, a gamma chain, two epsilon chains, and two zeta chains. The alpha and beta TCR chains include a variable (V) region and a constant (C) region. TCR diversity results from genetic recombination (VJ recombination of the alpha chain and VDJ recombination of the beta chain) that generates crossover areas that are important for antigen (ie peptide / MHC) recognition.

[0052] In some cases, the TCR repertoire library may include TCR-α chain sequences, TCR-β chain sequences, or both TCR-α and TCR-β chain sequences. The TCR chain sequences of the TCR repertoire libraries of the present disclosure may include full-length TCR chain sequences (e.g., full-length TCR alpha chain sequences, full-length TCR beta chain sequences) or partial TCR chain sequences (e.g., partial-length TCR alpha chain sequences, partial-length TCR beta chain sequences).

[0053] When a TCR repertoire library member of the disclosure comprises a partial TCR chain sequence, the partial TCR chain sequence may comprise the entire or essentially the entire TCR chain variable region (e.g., TCR alpha chain variable region, TCR beta chain variable region). In some cases, the resulting library member comprises a TCR variable region and at least a portion of a TCR constant region. In some cases, the resulting library member comprises a sequence corresponding to the TCR alpha and / or beta chain 5' mRNA end. In some cases, the resulting library member comprises a sequence derived from the TCR alpha or beta chain 5' end to at least a portion of the corresponding chain constant region.

[0054] In certain embodiments, the preparation of the immune cell receptor-specific library may include TCR-specific amplification. In such TCR-specific amplification, TCR-specific primers may be used. By "TCR-specific primer" is meant a primer that specifically hybridizes to a region of a nucleic acid sequence of a TCR chain (e.g., TCR alpha chain, TCR beta chain) or its complement. In some cases, the TCR-specific primer may hybridize to only one type of TCR chain, e.g., only the TCR alpha chain or only the TCR beta chain. In some cases, the TCR-specific primer may be configured to hybridize to more than one type of TCR chain, e.g., to the TCR alpha chain and the TCR beta chain. It may be configured to hybridize to both.

[0055] The TCR-specific primers may be designed to specifically hybridize to a TCR alpha chain constant region or its complement. For example, in some cases, the TCR-specific primers may hybridize to a mammalian TCR alpha chain constant region or its complement (including, for example, a human TCR alpha chain constant region, a mouse TCR alpha chain constant region, etc.).

[0056] An exemplary human TCR alpha chain constant region is PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 01) This sequence is CCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCA GAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA (SEQ ID NO: 02, T cell receptor alpha chain C region, human, GenBank: AY247834.1, AAO72258.1, UniProtKB: P01848) It is encoded by the nucleic acid sequence:

[0057] An exemplary mouse TCR alpha chain constant region is PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 03, UniProtKB: P01849) or PNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 04, GenBank: AAA53226.1) These sequences each have the amino acid sequences: CCATACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGC AATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTC CTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGT (SEQ ID NO: 05), CCAAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACC TGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGT (SEQ ID NO: 06, GenBank: U07662.1) It is encoded by the nucleic acid sequence:

[0058] The TCR-specific primers may be designed to specifically hybridize to a TCR beta chain constant region (e.g., TCR beta 1 or TCR beta 2) or its complement. For example, in some cases, the TCR-specific primers may hybridize to a mammalian TCR beta chain constant region or its complement (e.g., including a human TCR beta chain constant region, a mouse TCR beta chain constant region, etc.).

[0059] An exemplary human TCR beta chain 1 constant region is EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWT QDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 07, UniProtKB:P01850, GenBank:CAA25134.1) This sequence has the amino acid sequence: (SEQ ID NO: 08, GenBank: EF101778.1, X00437.1) It is encoded by the nucleic acid sequence:

[0060] An exemplary human TCR beta chain 2 constant region is DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 09, UniProtKB: A0A5B9, GenBank: AAA60662.1) This sequence has the amino acid sequence: (SEQ ID NO: 10, GenBank: L34740.1) It is encoded by the nucleic acid sequence:

[0061] An exemplary mouse TCR beta chain 1 constant region is EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 11, UniProtKB: P01852) This sequence has the amino acid sequence: (SEQ ID NO: 12, GenBank: FJ188408.1) It is encoded by the nucleic acid sequence:

[0062] An exemplary mouse TCR beta chain 2 constant region is EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 13, UniProtKB: P01851) This sequence has the amino acid sequence: (SEQ ID NO: 14, GenBank: U46841.1) It is encoded by the nucleic acid sequence:

[0063] In some cases, the immune cell receptor repertoire library may include a BCR repertoire library. The BCR complex is found on the surface of B cells and contains a membrane-bound immunoglobulin (i.e., antibody) binding portion, which contains a heavy chain and a light chain, each of which contains a constant (C) region and a variable (V) region. The immunoglobulin chains of the BCR are linked to the signaling CD79A / B chains by disulfide bridges. The immunoglobulin chains of the BCR are of various isotypes, including IgD, IgM, IgA, IgG, or IgE. Similar to TCRs, the immunoglobulin portion of the BCR undergoes V(D)J recombination, resulting in a very high degree of diversity within the population.

[0064] In some cases, the immune cell receptor repertoire library may include a BCR repertoire library, where, for example, the BCR repertoire library may include BCR immunoglobulin chain sequences (e.g., IgD, IgM, IgA, IgG, or IgE chain sequences). The immunoglobulin chain sequences of the BCR repertoire libraries of the present disclosure may include full-length immunoglobulin chain sequences (e.g., full-length heavy chain sequences, full-length light chain sequences) or partial immunoglobulin sequences (e.g., partial heavy chain sequences, partial light chain sequences).

[0065] When a BCR repertoire library member of the present disclosure comprises a partial immunoglobulin chain sequence, the partial immunoglobulin chain sequence may comprise an entire or essentially an entire immunoglobulin variable region (e.g., an immunoglobulin light chain variable region(s), an immunoglobulin heavy chain variable region(s)). In some cases, the resulting library member may comprise an immunoglobulin variable region(s) and at least a portion of an immunoglobulin constant region. In some cases, the resulting library member comprises a sequence corresponding to an immunoglobulin heavy and / or light chain 5' mRNA end. In some cases, the resulting library member comprises a sequence derived from the 5' end of an immunoglobulin heavy or light chain to at least a portion of a corresponding immunoglobulin chain constant region.

[0066] In certain embodiments, preparation of an immune cell receptor-specific library may include BCR-specific amplification (e.g., including immunoglobulin chain-specific amplification). In such immunoglobulin-specific amplification, immunoglobulin-specific primers may be used. By "immunoglobulin-specific primers" is meant primers that specifically hybridize to regions of an immunoglobulin chain (e.g., immunoglobulin heavy chain, immunoglobulin light chain) nucleic acid sequence or its complement. In some cases, immunoglobulin-specific primers may be used to specifically hybridize to regions of an immunoglobulin chain (e.g., immunoglobulin heavy chain, immunoglobulin light chain) nucleic acid sequence or its complement. It may hybridize only to globulin chains, for example only to heavy immunoglobulin chains, only to light immunoglobulin chains, only to IgD chains, only to IgM chains, only to IgA chains, only to IgG chains, only to IgE chains, and the like.

[0067] The immunoglobulin-specific primer may be designed to specifically hybridize to an immunoglobulin heavy chain constant region or its complement. For example, in some cases, the immunoglobulin-specific primer may hybridize to a mammalian immunoglobulin heavy chain constant region or its complement (including, for example, a human immunoglobulin heavy chain constant region, a mouse immunoglobulin heavy chain constant region, etc.).

[0068] The immunoglobulin-specific primer may be designed to specifically hybridize to an immunoglobulin light chain constant region or its complement. For example, in some cases, the immunoglobulin-specific primer may hybridize to a mammalian immunoglobulin light chain constant region or its complement (e.g., including a human immunoglobulin light chain constant region, a mouse immunoglobulin light chain constant region, etc.).

[0069] Amplification during library preparation (including, for example, immune receptor specific amplification) may be performed in a single round, or multiple rounds of amplification may be used. For example, in some cases, after a first round of amplification, one or more amplification primers not used in the first round may be added to the reaction mixture to facilitate a second round of amplification using the products of the first round of amplification as nucleic acid templates. In some cases, the second or subsequent round(s) of amplification may involve nested amplification, i.e., the primer binding sites used in the second or subsequent round(s) of amplification are located inside (i.e., one or more nucleotides from the 3' or 5' end) of the products generated in the first round of amplification. When nested amplification is used, the degree of nesting will vary as desired, for example, where the primer binding site in the second or third round or later is one or more (2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, etc.) nucleotides from the 3' or 5' end of the amplicon generated in the first round of amplification.

[0070] In some cases, the second or third or subsequent round(s) of amplification may not be nested, and the second round of amplification may use one or more primer binding sites used in the first round of amplification or primer binding sites added during the first round of amplification (e.g., primer binding sites added as part of a non-template sequence). In some cases, the second or third or subsequent rounds of amplification may use a nested primer amplification site at one end and a non-nested primer amplification site at the other end (e.g., a previously used or added primer binding site), and the nested site may be at the 3' end of the amplicon or the 5' end of the amplicon.

[0071] After certain library amplification steps, the prepared library may be considered ready for sequencing. In certain embodiments, the provided methods may further comprise subjecting the prepared immune cell receptor repertoire library to an NGS protocol. The protocol may be performed on any suitable NGS sequencing platform. Suitable NGS sequencing platforms include those provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ sequencing systems), those provided by Ion Torrent™ (e.g., Ion PGM™ and / or Ion PGM™), and those provided by the Ion Chromatography™ (Ion Chromatography™). The sequencing platform may include, but is not limited to, sequencing platforms provided by Biosciences (e.g., the PACBIO RS II Sequel Sequencing System), sequencing platforms provided by Life Technologies (e.g., the SOLiD Sequencing System), sequencing platforms provided by Roche (e.g., the 454 GS FLX+ and / or GS Junior Sequencing System), or any other suitable sequencing platform. NGS protocols will vary depending on the particular NGS sequencing system used. Detailed protocols for sequencing NGS libraries (which may include, for example, further amplification (e.g., solid-phase amplification), sequencing of amplicons, and analysis of sequencing data) are available from the manufacturer of the NGS sequencing system used.

[0072] Single Cells, Reaction Vessels and Droplets As outlined above, in some cases, RNA samples may be extracted from single cells to generate one or more libraries as described herein. Such "single cell libraries" may then be used in further downstream applications (such as sequencing applications). As used herein, "single cell" refers to one cell. Single cells useful as a source of template RNA and / or useful for generating single cell libraries (such as expression libraries and / or immune cell receptor repertoire libraries) can be taken from tissues of interest, or from biopsy specimens, blood samples, or cell cultures. Additionally, cells from specific organs, tissues, tumors, neoplasms, etc. can be taken and used in the methods described herein.

[0073] Single cells for use in such methods may be harvested by any convenient method. For example, in some cases, single cells may be harvested through limiting dilution of a cell sample. In some cases, the methods of the present disclosure may include a step of harvesting single cells. Single cell suspensions can be obtained using standard methods known in the art, including, for example, enzymatically using trypsin or papain to digest proteins binding cells in a tissue sample, detaching adherent cells in culture medium, or mechanically dissociating cells in a sample. Single cells can be placed in any suitable reaction vessel, where they can be individually processed. For example, a 96-well plate, a 384-well plate, or a plate with any number of wells (such as 2000, 4000, 6000, or 10000 or more). Multiwell plates can be part of a chip and / or an instrument. The present disclosure is not limited by the number of wells in a multiwell plate. In various embodiments, the total number of wells in the plate is between 100 and 200,000 or between 5000 and 10,000. In other embodiments, the plate includes smaller chips, each of which contains between 5,000 and 20,000 wells. For example, a square chip may contain 172 x 72 nanowells with a diameter of 0.1 mm.

[0074] In some cases, single cells may be obtained by sorting a cell sample using a cell sorter device. "Cell sorter," as used herein, refers to any device that allows for the sorting of individual cells into appropriate containers for downstream processing (such as library preparation processes as described herein).

[0075] Useful cell sorters include flow cytometers (such as those used in fluorescence activated cell sorting (FACS)). Flow cytometry is a method for sorting cells by the flow of liquid culture medium. Flow cytometry is a well-known technique that uses multi-parameter data to identify or distinguish different particle (e.g., cell) types, i.e., particles that differ from one another in terms of labeling (wavelength, intensity), size, etc., in the field. Flow cytometric analysis of a sample begins by placing an aliquot of the sample into a flow path of a flow cytometer. Within the flow path, cells in the sample pass through one or more sensing regions, essentially one at a time, where each cell is separately exposed to a single wavelength light source (or in some cases, two or more separate light sources), and measurements of scattering and / or fluorescence parameters are recorded separately for each cell, as desired. The data recorded for each cell is analyzed in real time or stored in a data storage and analysis means (such as a computer) for later analysis, as desired.

[0076] Cells sorted using a flow cytometer may be sorted into a common container (i.e., a single tube) or may be sorted separately into individual containers, for example, in some cases cells may be sorted into individual wells of a multi-well plate, as described below.

[0077] According to certain embodiments, cell sorting may include an upstream process of cell analysis and / or identification (sometimes referred to as phenotypic analysis). For example, in some cases, cells of a cell sample may be identified by FACS sorting as having a particular phenotypic characteristic (such as surface marker expression, viability, morphology, gene expression, cytokine expression, etc.) and selected for further processing based on that characteristic. For example, in some cases, cells of a cell sample may be sorted based on expression of one or more immune cell markers (e.g., including T cell markers, B cell markers, etc.) and collected for further downstream processing. In one example, T cells may be selected based on expression of one or more T cell surface markers (e.g., CD4, CD8, etc.) and the T cells may be collected for further processing. In some cases, cells collected (e.g., via FACS sorting) may be redistributed into a single cell sample prior to further processing (including library preparation, as described herein).

[0078] Useful cell sorters also include multi-well based systems that do not use flow cytometry. Such multi-well based systems include essentially any system that can place cells into individual wells of a multi-well container by any convenient means, including placing cells individually (e.g., by manually removing cells or dispensing with a robotic arm or pipettor) through the use of Poisson distribution (i.e., limiting dilution) statistics (e.g., multi-sample nanodispense (MSND) system). In some cases, useful multi-well systems include multi-well wafers or chips, where cells are placed into wells or wafers / chips and individually identified by a microscopic analysis system. In some cases, an automated microscopic analysis system may be used in conjunction with a multi-well wafer / chip to automatically identify individual cells for downstream analysis (including library preparation, as described herein).

[0079] In some cases, one or more cells may be sorted or otherwise transferred to a suitable reaction vessel, including vessels sufficient to perform one or more aspects of library preparation as described herein. Reaction components may be added to the reaction vessel, including, for example, components for preparing an RNA sample, components for generating product double-stranded cDNA, components for one or more library preparation reactions, etc. There are a variety of reaction vessels that can contain the reaction mixture and its components and in which the reactions of the disclosed methods can be performed. Useful reaction vessels include, for example, tubes (e.g., single tubes, multi-tube strips, etc.), wells (e.g., multi-well plates (e.g., 96-well plates, 384-well plates, etc.), and the like. , or any number of wells (such as 2000, 4000, 6000, or 10000 or more). Multi-well plates can be freestanding or part of a chip and / or device.

[0080] In some cases, the reaction mixture and its components may be added to liquid droplets (e.g., oil-in-water emulsion droplets), for example, as described in more detail below, and the reactions of the disclosed methods may be carried out in liquid droplets (e.g., water-in-oil emulsion droplets). Although droplets can serve the purpose of individual reaction vessels, the droplets (or emulsions containing droplets) will generally be placed in suitable vessels (e.g., tubes, wells, or microfluidic channels, etc.). Amplification reactions carried out in droplets may be sorted, for example, based on fluorescence (e.g., fluorescence from nucleic acid detection reagents or labeled probes) using a fluorescence-based droplet sorter. Useful fluorescence-based droplet sorters are numerous and may include, for example, flow cytometers, microfluidic-based droplet sorters, etc.

[0081] As described above, in protocols that include a pooling step, the pooling step can be performed after the generation of product double-stranded cDNA, for example, from single cells, droplets, wells, etc. Thus, in certain embodiments of the methods described herein, cells are obtained from the tissue of interest (e.g., blood) to obtain a single cell suspension. A single cell is placed in one of the wells of a multi-well plate or other suitable container (such as a microfluidic chamber or tube). The cells are lysed and reaction components are added directly to the lysate. Whether or not pooling of single cell samples is used, the generated library may be sequenced to generate reads. This allows the genes expressed in each single cell to be identified.

[0082] In certain embodiments of the methods described herein, droplets are obtained and a single droplet is sorted into one of the wells of a multi-well plate or other suitable container (such as a microfluidic chamber or tube). A reaction mixture may be added directly to the droplet, for example, without further purification.

[0083] In some cases, the method of the present disclosure may include obtaining a single droplet. Obtaining droplet cells may be performed according to any convenient protocol, including, for example, mechanically sorting the droplets (e.g., using a fluorescence-based sorter, such as a flow cytometer or a microfluidic-based sorter). The single droplets may be placed in any suitable reaction vessel that allows for individual processing of the single droplets. For example, a 96-well plate, a 384-well plate, or a plate with any number of wells, such as 2000, 4000, 6000, or 10000 or more. The multi-well plate may be part of a chip and / or an instrument. The present disclosure is not limited by the number of wells in the multi-well plate. In various embodiments, the total number of wells in the plate is between 100 and 200,000 or between 5000 and 10,000. In another embodiment, the plate includes smaller chips, each of which includes between 5,000 and 20,000 wells. For example, a square chip may contain 72 x 72 or 125 x 125 nanowells with a diameter of 0.1 mm.

[0084] The wells (e.g., nanowells) of a multiwell plate may be made of any convenient size, shape, or volume. The wells may be 100 μm to 1 mm long, 100 μm to 1 mm wide, and 100 μm to 1 mm deep. In various embodiments, each nanowell has an aspect ratio (ratio of depth to width) of 1 to 4. In one embodiment, each nanowell has an aspect ratio of 2. The cross-section may be circular, elliptical, ovoid, conical, rectangular, triangular, It may be polyhedral or of any other shape. The cross-section of the wells at any given depth may vary in size and shape.

[0085] In certain embodiments, the wells have a volume between 0.1 nl and 1 μl. The nanowells may have a volume of 1 μl or less (e.g., 500 nl or less). The volume may be 200 nl or less (e.g., 100 nl or less). In embodiments, the nanowells have a volume of 100 nl. If desired, the nanowells can be fabricated to increase the surface area to volume ratio, thereby promoting heat transfer through the unit and thereby reducing the ramp time of thermal cycling. The holes in each well (e.g., nanowells) can take on a variety of shapes. For example, the holes in the wells can be separated by straight or curved walls to form separate adjacent compartments, or separated by annular walls to form an inner annular compartment and an outer annular compartment.

[0086] The wells can be designed to accommodate a single cell or droplet per well. Individual cells or droplets can be separated in any other suitable container, such as a microfluidic chamber, droplet, nanowell, tube, etc. Any convenient method of manipulating single cells or droplets can be used, including the use of fluorescence activated cell sorting (FACS), robotic device injection, gravity flow or micromanipulation, and semi-automated cell pickers (e.g., Stoelting Co.'s Quixell™ cell transfer system), etc. In some cases, single cells or droplets can be placed into the wells of the plate according to Poisson statistics (e.g., about 10%, 20%, 30%, or 40% or more of the wells contain single cells or droplets (this value can be defined by adjusting the number of cells or droplets in a given unit volume of fluid dispensed into the container)). In some cases, the suitable reaction container contains droplets (e.g., microdroplets). Individual cells or droplets can be individually selected, for example, based on characteristics detectable by microscopy (location, morphology, presence (e.g., expression) of a reporter gene, presence of bound antibody (e.g., antibody labeling), FISH, presence of RNA (e.g., intracellular RNA labeling), etc.) or qPCR.

[0087] For example, after obtaining a desired cell population or single cells as described above, nucleic acids can be released from the cells by lysing the cells. Lysis can be performed, for example, by heating or freezing and thawing the cells, by using detergents or other chemical methods, or by a combination of these. However, any suitable lysis method can be used. In some cases, a gentle lysis procedure can be used to advantage to prevent the release of nuclear chromatin, thereby avoiding genomic contamination of the cDNA library and minimizing degradation of mRNA. For example, heating the cells at 72°C for 2 minutes in the presence of Tween-20 is sufficient to lyse the cells, with no detectable genomic contamination from the nuclear chromatin. Alternatively, cells can be heated to 65°C for 10 minutes in water (Esumi et al., Neurosci Res 60(4):439-51 (2008)) or 70°C for 90 seconds in PCR buffer II (Applied Biosystems) supplemented with 0.5% NP-40 (Kurimoto et al., Nucleic Acid Res 34(5):e42 (2006)), or lysis can be performed with a protease (such as Proteinase K) or by using a chaotropic salt (such as guanidine isothiocyanate) (U.S. Patent Application Publication No. 2007 / 0281313).

[0088] If desired, a given single cell or droplet workflow may include a pooling step, in which a nucleic acid product composition comprised of, for example, product double-stranded cDNA is pooled in one or more of the following: The nucleic acid product compositions are combined or pooled with nucleic acid product compositions from one or more additional cells or droplets. The number of different nucleic acid product compositions made from different cells or droplets that are combined or pooled in such embodiments can vary, in some cases ranging from 2 to 50, such as 3 to 25, including 4 to 20, or even 10,000 or more.

[0089] In some embodiments, a multi-sample nanodispenser (MSND) system is used, which includes a multi-well plate, for example in the form of an addressable nanowell array, and a sample dispenser. An example of such an MSND system is the ICELL8® Single-Cell MSND System (Wafergen, Fremont, Ca). Further details of the ICELL8® MSND system can be found in U.S. Patent Nos. 7,833,709 and 8,252,581, and U.S. Patent Application Publication Nos. 2015 / 0362420 and 2016 / 0245813, the disclosures of which are incorporated herein by reference.

[0090] Tagmentation As outlined above, in some cases, the provided method may include end-capturing one or more ends of the product double-stranded cDNA produced.In certain embodiments, such end-capturing may utilize tagmentation reaction, and this reaction may use one or more tagmentation reaction components.The tagmentation process used may be various, and may include, for example, 5' end capture by tagmentation reaction, 3' end capture by tagmentation reaction, or a combination thereof.

[0091] When tagmentation is present in the provided methods, the transposome used in tagmentation may comprise a transposase and a transposon nucleic acid (including a transposon end domain and a post-tagmentation PCR primer binding domain). These domains are functionally defined and may be domains within the same sequence or of different sequences, depending on the needs of the researcher. These domains may overlap, such that a portion of the post-tagmentation PCR primer binding domain is present in the transposon end domain.

[0092] "Transposase" refers to an enzyme that can form a functional complex with a transposon end domain-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and catalyze the insertion or transposition of the transposon end-containing composition into double-stranded target DNA with which it is incubated in an in vitro transposition reaction. Transposases that are useful in practicing the provided methods include, but are not limited to, Tn5 transposase, Tn7 transposase, and Mu transposase. The transposase can be a wild-type transposase. In another aspect, the transposase contains one or more modifications (e.g., amino acid substitutions) to improve a property of the transposase, e.g., to increase the activity of the transposase. For example, hyperactive mutants of Tn5 transposase have been developed that have substitution mutations in the Tn5 protein (e.g., E54K, M56A, and L372P), see, e.g., Picelli et al. al. (2013) Genome Research 24:2033-2040.

[0093] The term "transposon end domain" refers to double-stranded DNA consisting only of a nucleotide sequence ("transposon end sequence") that is necessary for the formation of a complex with a transposase or integrase enzyme that is functional in an in vitro transposition reaction. A transposon end domain recognizes and binds to the transposon end domain. With the bound transposase or integrase, it forms a "complex," "synaptic complex," "transposome complex," or "transposome composition," which is capable of inserting or transposing its transposon end domain into a target DNA incubated with the complex in an in vitro transposition reaction. The transposon end domain exhibits two complementary sequences, the "imported transposon end sequence" or "imported strand" and the "non-imported transposon end sequence" or "non-imported strand." For example, one transposon end domain that forms a complex with a hyperactive Tn5 transposase (e.g., EZ-Tn5 transposase, EPICENTRE Biotechnologies, Madison, Wis., USA) that is active in an in vitro transposition reaction comprises an import strand that exhibits an "import transposon end sequence" of 5'AGATGTGTATAAGAGACAG3' (SEQ ID NO: 15) and a non-import strand that exhibits a "non-import transposon end sequence" of 5'CTGTCTCTTATACACATCT3' (SEQ ID NO: 16). In an in vitro transposition reaction, the 3' end of the import strand is joined or transferred to the target DNA. In an in vitro transposition reaction, the non-import strand that exhibits a transposon end sequence that is complementary to the import transposon end sequence is not joined or transferred to the target DNA. The sequence of the particular transposon end domain used in practicing the provided methods will vary depending on the particular transposase used. For example, when used in conjunction with a Tn5 transposase, a Tn5 transposon end domain may be included in the transposon nucleic acid. Further details regarding transposases and transposon end domains that may be used in the transposomes of the present invention include, but are not limited to, those described in U.S. Patent Nos. 9,040,256, 9,080,211, 9,080,211, and 9,115,396, the disclosures of which are incorporated herein by reference.

[0094] In addition to the transposon end domain, the transposon nucleic acid may also include a post-tagmentation primer binding domain. In some cases, the post-tagmentation primer binding domain may later be used in an amplification reaction to add a sequencing platform adapter construct domain (e.g., through the use of a primer that hybridizes to the post-tagmentation primer binding domain and has a sequencing platform adapter construct domain bound thereto). In some cases, the post-tagmentation primer binding domain may include a sequencing platform adapter construct domain.

[0095] The sequencing platform adaptor construct domains added during tagmentation or amplification (which follows and is dependent on tagmentation) will vary. Such sequencing platform adaptor constructs can be a nucleic acid domain selected from a domain (e.g., a "capture site" or "capture sequence") that specifically binds to a surface-attached sequencing platform oligonucleotide (e.g., a P5 or P7 oligonucleotide attached to the surface of a flow cell of an Illumina® sequencing system), a sequencing primer binding domain (e.g., a domain to which the Read1 or Read2 primer of the Illumina® platform can bind), a barcode domain (e.g., a domain that uniquely identifies the sample source of the nucleic acid being sequenced, allowing multiplexing of samples by marking all molecules from a given sample with a specific barcode or "tag"), a barcode sequencing primer binding domain (a domain to which a primer used in sequencing the barcode binds), a molecular specific domain, or any combination of such domains.

[0096] As shown in FIG. 4, a transposase and a transposon nucleic acid (including a transposon end domain and a second post-tagmentation PCR primer binding domain) The product double-stranded cDNA is tagmented using a transposome containing Tn5 transposase and Illumina® Nextera® TnRP1 or TnRP2 sequences (Figure 4).

[0097] It can be seen that various variations of the exemplary end capture method via tagmentation shown in FIG. 4 are possible. Instead of capturing the 3' end of the RNA, for example, the method can be used to capture the 5' end of the RNA. 5' end capture can be achieved, for example, by introducing a post-tagmentation PCR primer binding domain (e.g., RP2 sequence) into the template switch oligonucleotide instead of into the first strand cDNA primer. According to this variation, post-tagmentation amplification can be performed using an amplification primer that binds to the post-tagmentation PCR primer binding domain originally present in the template switch oligonucleotide, in conjunction with an amplification primer that binds to the TnRP1 or TnRP2 sequence added during the tagmentation step.

[0098] In another variation, rather than using two types of transposomes (such as the TnRP1 or TnRP2 transposomes used in the above examples), one type of transposome (a transposome with one type of post-tagmentation PCR primer binding domain) can be used. Amplification of the desired tagmentation product can be performed using a primer that binds to one type of post-tagmentation PCR primer binding domain provided by the transposome in conjunction with a primer that binds to the post-tagmentation PCR primer binding domain added in a previous step (such as first strand synthesis or amplification of the double-stranded product nucleic acid).

[0099] As a non-limiting example, the product double-stranded cDNA may be split into two reactions and then tagmented with the product double-stranded cDNA, resulting in the introduction of a TnRP1 sequence at the tagmented 5' end of the product double-stranded cDNA. An embodiment of this example is shown in FIG. 5, which is described in more detail below. As shown in FIG. 5, a transposon sequence (e.g., "TnRP1") is added to the 3' end of the captured 5' product double-stranded cDNA by tagmentation. In a subsequent step, the added transposon sequence is used as a primer binding site during amplification of the captured 5' product double-stranded cDNA. Specifically, in the illustrated example, a first primer ("5' end capture primer 1") that hybridizes to the sequence added in the template switching reaction and a second primer ("5' end capture primer 2") that binds to the "Tn Read1" sequence are used to amplify the captured 5' product double-stranded cDNA while simultaneously adding the sequencing platform adaptor construct domains required for library preparation for NGS.

[0100] In some cases, the primer that binds to the introduced transposon sequence, for example at the newly created double-stranded nucleic acid end due to the nuclease activity of the transposase, may be referred to as a terminal amplification primer. In this context, such terminal amplification primers may be used to amplify from the end created by tagmentation, for example toward the original end of the RNA of interest. For example, in some cases, the amplification reaction may use a terminal amplification primer and a second primer that amplifies from the 5' end of the double-stranded cDNA (i.e., the end corresponding to the original 5' end of the RNA). In some cases, the amplification reaction may use a terminal amplification primer and a second primer that amplifies from the 3' end of the double-stranded cDNA (i.e., the end corresponding to the original 3' end of the RNA).

[0101] Other variations include, for example, replacing the Illumina®-specific sequencing domains in the various primers / oligonucleotides with the sequencing domains required for, for example, Ion Torrent™ sequencing systems (e.g., Ion PGM™ and Ion Proton™ sequencing systems), Pacific Biosciences sequencing systems (e.g., the PACBIO RS II sequencing system), Life Technologies™ sequencing systems (e.g., the SOLiD sequencing system), Roche sequencing systems (e.g., the 454 GS FLX+ and GS Junior sequencing systems), or any other applicable sequencing platform.

[0102] In a further variation, tagmentation may use three or more types of transposomes, rather than using one or two types of transposomes (such as TnRP1 transposomes or TnRP2 transposomes used in the above examples). For example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, fifty or more, or one hundred or more transposomes with different post-tagmentation PCR primer binding domains may be used. The tagmentation product of interest in such tagmented samples may be amplified using a primer that binds to the post-tagmentation PCR primer binding domain of a particular type of transposome, in conjunction with the primer that binds to the post-tagmentation PCR primer binding domain added in a previous step (such as synthesis or amplification of the first strand of double-stranded product nucleic acid).

[0103] If it is desired to prepare transposomes for the tagmentation step, any suitable transposome preparation approach may be used, and such approaches may vary, for example, depending on the specific transposase and transposon nucleic acid used. For example, the transposon nucleic acid and the transposase may be incubated together in a suitable buffer at a suitable molar ratio (e.g., 2:1 molar ratio, 1:1 molar ratio, 1:2 molar ratio, etc.). According to one embodiment, when the transposase is a Tn5 transposase, the preparation of the transposome may include incubating the transposase and the transposon nucleic acid in a 1:1 molar ratio in 2xTn5 dialysis buffer for a sufficient period of time (e.g., 1 hour).

[0104] Tagmentation of the product double-stranded cDNA involves contacting the double-stranded cDNA with transposomes under tagmentation conditions. Such conditions may vary depending on the particular transposase used. Typically, the conditions will include incubating the transposomes and tagged extension products in a buffered reaction mixture (e.g., a reaction mixture buffered with Tris acetate) at a pH of 7-8 (e.g., pH 7.5). The transposomes may be provided such that they are present in about equimolar amounts or in molar excess of transposon compared to the tagged extension products. Suitable temperatures include 32°C to 42°C (e.g., 37°C). The reaction is allowed to proceed for a sufficient period of time (e.g., 5 minutes to 3 hours). The reaction may be stopped by adding a solution (e.g., a "stop" solution) that may contain an amount of SDS and / or other transposase reaction stopping reagent suitable for stopping the reaction. Protocols and materials for fragmenting nucleic acids using transposomes are available, such as those provided in the EZ-Tn5™ Transposome Kit available from EPICENTRE Biotechnologies (Madison, Wis., USA).

[0105] Then, one or more post-tagmentation PCR primers that hybridize to the one or more post-tagmentation primer binding sites added during the tagmentation reaction. The resulting tagmented sample may be subjected to PCR amplification conditions using a post-tagmentation primer. The post-tagmentation primer may comprise a sequencing platform adaptor domain. The sequencing platform adaptor construct(s) may comprise any of the nucleic acid domains described elsewhere herein (e.g., a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular specific domain, or any combination thereof). Such embodiments are useful, for example, when the nucleic acid of the tagmented sample does not contain all of the adaptor domains useful or necessary for sequencing on the relevant sequencing platform, and the remaining adaptor domains are provided by the primers used to amplify the nucleic acid of the tagmented sample.

[0106] Template switching reverse transcription As outlined above, aspects of the disclosed methods may involve the use of template switching reverse transcription. For example, in some cases, the disclosed methods may include using template switching reverse transcription to generate product double-stranded cDNA from a nucleic acid sample. Thus, in some cases, template switching reverse transcription may be used to generate double-stranded cDNA from a template nucleic acid, and then the double-stranded cDNA may be split into separate reaction mixtures that can be used separately to prepare one or more libraries (such as an expression library and / or an immune cell receptor repertoire library).

[0107] A template switching reverse transcription reaction will generally involve a template nucleic acid from which the product double stranded cDNA is generated. There are various sources and / or methods for generating the template nucleic acid. The template nucleic acid may be present in a template nucleic acid composition (e.g., a defined composition) or in a biological sample (e.g., a sample taken from or including an organism and / or living cell). The biological sample containing the template nucleic acid may be prepared by any convenient means to make the nucleic acid of the sample available to the components (e.g., primers, oligonucleotides, etc.) of the methods described herein.

[0108] There are various methods for preparing a biological sample containing template nucleic acid. Useful processes may include, but are not limited to, homogenizing the sample, lysing one or more cell types of the sample, concentrating the desired nucleic acid of the sample, removing one or more components present in the sample (e.g., proteins, lipids, contaminating nucleic acids), and performing nucleic acid isolation to isolate the template nucleic acid. In some cases, the cells of the biological sample may be prepared by lysing the cells of the sample. Useful processes for lysing cells include, but are not limited to, chemical lysis, enzymatic lysis, mechanical lysis, freeze / thaw lysis, and the like. In some cases, the cells of the sample may not be fixed before using the template nucleic acid obtained from a plurality of cells or a single cell of the sample in a method as described herein. In some cases, the cells of the sample may be fixed before using the template nucleic acid obtained from a plurality of cells or a single cell of the sample in a method as described herein.

[0109] The template nucleic acid of the present disclosure may include multiple distinct template nucleic acids with different sequences. The template nucleic acid (e.g., template RNA, template DNA, etc.) may be a polymer of any length. The length of the polymer may vary, but in some cases, the polymer is 10 nt or more, 20 nt or more, 50 nt or more, 100 nt or more, 500 nt or more, 1000 nt or more, 2000 nt or more, 3000 nt or more. , 4000 nt or more, 5000 nt or more or more. In certain embodiments, the template nucleic acid is a polymer, and the number of bases in the polymer can vary, and in some cases is 10 nt or less, 20 nt or less, 50 nt or less, 100 nt or less, 500 nt or less, 1000 nt or less, 2000 nt or less, 3000 nt or less, 4000 nt or less, 5000 nt or less, 10,000 nt or less, 25,000 nt or less, 50,000 nt or less, 75,000 nt or less, 100,000 nt or less.

[0110] According to a particular embodiment, the template nucleic acid is a template ribonucleic acid (template RNA). The template RNA may be any type of RNA (or subtype thereof), including, but not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting small interfering RNA (ta-siRNA), naturally occurring small interfering RNA (nat-siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), transfer-messenger RNA (tmRNA), precursor messenger RNA (pre-mRNA), Cajal body-specific small RNA (scaRNA), piwi-interacting RNA (piRNA), endoribonuclease-prepared siRNA (esiRNA), transient small RNA (stRNA), signal recognition RNA, telomeric RNA, ribozyme, or any combination of these types of RNA or their subtypes.

[0111] The number of distinct template nucleic acids with different sequences in a given template nucleic acid composition can vary. The number of distinct template nucleic acids in a given template nucleic acid composition can vary, but in some cases the number of distinct template nucleic acids in a given template nucleic acid composition can be between 1 and 10. 8 pieces (1~10 7 Pieces etc. (1~10 5 The range is inclusive).

[0112] The template nucleic acid composition used in the methods as described above may be any suitable nucleic acid sample. The nucleic acid sample containing the template nucleic acid may be combined with a reaction mixture in an amount sufficient to generate product nucleic acid. According to one embodiment, the nucleic acid sample is combined with the reaction mixture such that the final concentration of nucleic acid in the reaction mixture is between 1 fg / μL and 10 μg / μL (1 pg / μL to 5 μg / μL, 0.001 μg / μL to 2.5 μg / μL, 0.005 μg / μL to 1 μg / μL, 0.01 μg / μL to 0.5 μg / μL, etc. (including 0.1 μg / μL to 0.25 μg / μL)). In certain aspects, the nucleic acid sample containing the template nucleic acid is isolated from a single cell, for example, as described in more detail below. In another aspect, the nucleic acid sample comprising the template nucleic acid is isolated from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 20 or more, 50 or more, 100 or more, or 500 or more cells. According to certain embodiments, the nucleic acid sample comprising the template nucleic acid is isolated from 500 or less, 100 or less, 50 or less, 20 or less, 10 or less, 9, 8, 7, 6, 5, 4, 3, or 2 cells.

[0113] The template nucleic acid may be present in any nucleic acid sample of interest, including, but not limited to, a nucleic acid sample isolated from a single cell, a plurality of cells (e.g., cultured cells), a tissue, an organ, or an organism (e.g., mouse, rat, etc.). In certain aspects, the nucleic acid sample is isolated from a cell(s), tissue, organ, and / or the like of a mammalian animal (e.g., a human, a rodent (e.g., a mouse), or any other mammalian animal of interest). In other aspects, the nucleic acid sample is isolated from a source other than a mammalian animal, such as an amphibian animal (e.g., a frog (e.g., Xenopus), a fish (zebrafish (Danio rerio)), or any other source of a nucleic acid sample other than a mammalian animal).

[0114] Approaches, reagents and kits for isolating nucleic acids from such sources are known in the art. For example, kits for isolating nucleic acids from a source of interest are commercially available (such as NucleoSpin®, NucleoMag® and NucleoBond® genomic DNA or RNA isolation kits from Takara Bio USA, Inc. (Mountain View, CA)). In certain embodiments, nucleic acids are isolated from fixed biological samples, such as formalin-fixed, paraffin-embedded (FFPE) tissues. Nucleic acids from FFPE tissues may be isolated using commercially available kits (such as NucleoSpin® FFPE DNA or RNA isolation kits from Takara Bio USA, Inc. (Mountain View, CA)).

[0115] A general depiction of a template switching reaction is shown in FIG. 3. In the example shown, a single product nucleic acid primer (300) hybridizes to a template nucleic acid (301) through a complementary sequence (denoted by "XXXX") shared by the single product nucleic acid primer and the template. The single product nucleic acid primer may, but need not, include a region of additional sequence (302) that is not complementary to the template (e.g., a non-template region). After annealing the single product nucleic acid primer to the template, reverse transcription (303) proceeds by use of a reverse transcriptase to generate a single product nucleic acid strand (304) that is complementary to the template. The reverse transcriptase with terminal transferase activity transfers the non-template nucleotides (denoted by "YYY") to the generated single product nucleic acid, and a template switching oligonucleotide (305) hybridizes to the non-template nucleotides of the single product nucleic acid through a sequence of complementary nucleotides (also denoted by "YYY" and also referred to herein as the 3' hybridization domain) present in the template switch oligonucleotide. The template switch oligonucleotide includes an additional sequence (306) that does not hybridize to the non-template nucleotide. Template switching occurs (307) and the reverse transcriptase switches from the template and uses the template switching oligonucleotide as a second template to transcribe the additional sequence (306) to generate its complement (308). The single product nucleic acid strand thus generated includes the complete sequence of the single product nucleic acid primer including any additional sequence (302) that was not hybridized to the template, if any, the complementary sequence of the template, and the complementary sequence of the template switch oligonucleotide. Methods and reagents related to template switching are also described in U.S. Pat. No. 9,410,173, the disclosure of which is incorporated herein by reference in its entirety.

[0116] Template Switching Oligonucleotides In a template switching reverse transcription reaction, a template switch oligonucleotide may be used. By "template switch oligonucleotide" is meant an oligonucleotide template to which a polymerase switches from an initial template (e.g., a template nucleic acid (e.g., an RNA template)) during a nucleic acid polymerization reaction. In this context, the template may be referred to as a "donor template" and the template switch oligonucleotide may be referred to as an "acceptor template." As used herein, an "oligonucleotide" is a single-stranded nucleotide multimer of 2-500 nucleotides, e.g., 2-200 nucleotides. The oligonucleotide may be synthetic or enzymatically produced, and in some embodiments, is 10-50 nucleotides in length. The oligonucleotide may comprise ribonucleotide monomers (i.e., an oligoribonucleotide or "RNA oligonucleotide") or deoxyribonucleotide monomers (i.e., an oligodeoxyribonucleotide or "DNA oligonucleotide"). The oligonucleotide may be, for example, 10-20 nucleotides, 21-30 nucleotides, 31-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides, 100-120 nucleotides, 130-140 nucleotides, 140-160 nucleotides, 170-180 nucleotides, 190-200 nucleotides, 21-22 nucleotides, 22-24 nucleotides, 25-26 nucleotides, 26-28 nucleotides, 27-30 nucleotides, 28-32 nucleotides, 32-36 nucleotides, 36-38 nucleotides, 37-39 nucleotides, 38-40 nucleotides, 39-40 nucleotides, 40- It may be 40 nucleotides, 41-50 nucleotides, 51-60 nucleotides, 61-70 nucleotides, 71-80 nucleotides, 80-100 nucleotides, 100-150 nucleotides, or 150-200 nucleotides in length, up to a maximum of 500 nucleotides or more in length.

[0117] In a template switching reverse transcription reaction, a suitable reaction mixture may be used. A suitable reaction mixture for a template switching reverse transcription reaction may include the template switch oligonucleotide at a concentration sufficient to facilitate template switching of the polymerase from the template to the template switch oligonucleotide and further facilitate extension by the polymerase using any additional sequences of the template switch oligonucleotide as a template, if any. For example, the template switch oligonucleotide may be added to the reaction mixture at a final concentration of 0.01 to 100 μM (such as 0.1 to 10 μM, such as 0.5 to 5 μM, including 1 to 2 μM (e.g., 1.2 μM)).

[0118] In a template switching reverse transcription reaction, the template switch oligonucleotide may or may not contain one or more nucleotides (or analogs thereof) that are modified or otherwise unnatural. For example, the template switch oligonucleotide may contain one or more nucleotide analogs (e.g., LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, etc.), linkage modifications (e.g., phosphorothioates, 3'-3' reverse conjugates, 5'-5' reverse conjugates), 5' and / or 3' end modifications (e.g., 5' and / or 3' amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides the template switch oligonucleotide with a desired function.

[0119] In certain embodiments, the template switch oligonucleotide comprises a 3' hybridization domain. The 3' hybridization domain may vary in length, in some cases ranging from 2-10 nt in length, such as 3-7 nt in length. The 3' hybridization domain of the template switch oligonucleotide may comprise a sequence that is complementary to a non-template sequence that is added to a single product nucleic acid (e.g., cDNA) of the template switching reaction. A non-template sequence, as described in more detail below, generally refers to a sequence that does not correspond to and is not template-templated, such as an RNA template or a DNA template. The non-template sequence, when present in the 3' hybridization domain of the template switch oligonucleotide, may encompass the entire 3' hybridization domain or a portion thereof. In some cases, the non-template sequence may comprise or consist of a heteropolynucleotide, which may vary in length from 2 nt to 10 nt, such as 3-7 nt in length, including 3 nt. In some cases, the non-template sequence may comprise or consist of a homopolynucleotide, which may vary in length from 2 nt to 10 nt, such as from 3 to 7 nt in length, inclusive.

[0120] The template switch oligonucleotide can be free in solution or bound to a solid support (e.g., a bead). In some cases, the template switch oligonucleotide is dried in a container (e.g., a multiwell array chip). The dried template switch oligonucleotide can be covalently or non-covalently bound to the container.

[0121] Tail sequence and tailing In some cases, the disclosed method may include generating a double-stranded product cDNA and / or amplifying a template nucleic acid with a primer having a sequence that is complementary to the tail sequence of the template nucleic acid having a tail sequence. The term "tail sequence" as used herein generally refers to a polynucleotide region at the 3' end of a template nucleic acid that is composed of one type of nucleotide species (e.g., A, C, G, T, etc.). In some cases, a first strand complementary deoxyribonucleic acid (cDNA) primer may be complementary in whole or in part to a tail sequence. For example, a poly(A) tail of an mRNA template is one non-limiting example of a tail sequence. Thus, a first strand cDNA primer may in some cases include or consist of a poly(T) sequence that is complementary to a poly(A) tail of an mRNA template.

[0122] Tail sequences may be naturally present in a template nucleic acid of the present disclosure or may be synthetically added. Thus, examples of tail sequences that may be present in a template nucleic acid of the present invention include, but are not limited to, a poly(A) tail, a poly(C) tail, a poly(G) tail, a poly(T) tail, etc. The size of the tail sequence may range from less than 10 nt to 300 nt or more, and examples thereof include, but are not limited to, 10 to 300 nt, 10 to 200 nt, 10 to 150 nt, 10 to 100 nt, 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 40 nt, 10 to 30 nt, 10 to 20 nt, 20 to 300 nt, 20 to 200 nt, 20 to 150 nt, 20 to 100 nt, 20 to 90 nt, 20 to 80 nt, 20 to 70 nt, 20 to 60 nt, 20 to 50 nt, 20 to 40 nt, 20 to 30 nt, 15 nt, 16 nt, 18 nt, and 20 nt. When the template nucleic acid includes a tail sequence, a primer used to generate the double-stranded product cDNA, e.g., a first strand cDNA primer, may include a sequence that is complementary to the tail sequence to which the primer hybridizes and primes the extension of the first strand cDNA. Useful sequences that are complementary to the tail sequence are numerous and may include, but are not limited to, poly(dA), poly(dC), poly(dG), poly(dT), and the like.

[0123] As discussed above, the tail sequence present in the template nucleic acid may be natural (e.g., in the case of a poly(A) tail of an mRNA template) or may be artificially or synthetically generated. For example, in some cases, the tail sequence may be added to the nucleic acid template in a tailing reaction. There are various tailing reactions, which may include, for example, adding the tail sequence to the template through an enzymatic process. Enzymes useful for adding tails to the nucleic acid templates of the present disclosure include, but are not limited to, terminal transferases (e.g., terminal deoxynucleotidyl transferase, RNA-specific nucleotidyl transferase, etc.). The nucleotide species of the tailing sequence may be controlled as desired, for example, by having only the desired type of dNTPs available in the tailing reaction using the terminal transferase (e.g., only dATP, only dCTP, only dGTP, or only dTTP). In some cases, a "dNTP tailing mix" is used in the tailing reaction, such mix containing only one type of dNTP (e.g., ATP). In some cases, the nucleic acid template may be prepared for the tailing reaction, for example, by removing (dephosphorylating) the 3' phosphate present in the nucleic acid template. Any convenient and suitable phosphatase may be used for such purposes, including, but not limited to, alkaline phosphatase (e.g., shrimp alkaline phosphatase and its derivatives).

[0124] In some cases, the disclosed methods include providing conditions, e.g., in the presence of dNTP species, sufficient to generate a template having a tail sequence (i.e., a tailed template). The method may include performing a tailing reaction by contacting the template nucleic acid with terminal transferase under a low temperature to add a tailing sequence to the template nucleic acid. The dNTP addition rate, i.e., the length of the tail sequence, is derived from the ratio of the 3' end to the dNTP concentration and which dNTP is used. The terminal transferase reaction is performed at a temperature at which the terminal transferase is active (such as 30°C to 50°C (including 37°C)). The dNTPs in the terminal transferase reaction may be present at a final concentration of 0.01mM to 1mM (such as 0.05mM to 0.5mM (including 0.1mM)). The template nucleic acid may be present in the terminal transferase reaction at a concentration of 0.05 to 500pmol (0.5 to 50pmol (including 1 to 25pmol), for example, 5pmol). A terminal transferase buffer solution and any other useful components (e.g., metal cofactors such as Co) may also be included in the terminal transferase reaction, for example, as a separate solution (e.g., buffer) or as part of a "dNTP tailing mix." The terminal transferase reaction adds nucleotides to the 3' end of the nucleic acid template, and the resulting tailed template nucleic acid may then be used in further steps of the reaction according to the disclosed methods.

[0125] In some cases, the template switch oligonucleotide contains a modification that prevents the polymerase from switching from the template switch oligonucleotide to a different template nucleic acid after synthesizing the complement of the 5' end of the template switch oligonucleotide (e.g., the 5' adapter sequence of the template switch oligonucleotide). Useful modifications include, but are not limited to, abasic lesions (e.g., tetrahydrofuran derivatives), nucleotide adducts, and isonucleotide bases (e.g., isocytosine, isoguanine, and / or the like), and any combination thereof.

[0126] In some cases, the template switch oligonucleotide may include a 5' adapter sequence (e.g., a defined nucleotide sequence 5' of the 3' hybridization domain of the template switch oligonucleotide), which may serve various purposes in downstream applications. In some cases, the 5' adapter sequence may serve as a primer binding site for further amplification of the amplified dsDNA, or for example, nested amplification or suppression amplification.

[0127] Primer A single-product nucleic acid primer (also referred to as a single-product nucleic acid synthesis primer (e.g., first strand cDNA primer) or first strand primer) comprises a template binding domain. For example, the nucleic acid may comprise a first (e.g., 3') domain that is configured to hybridize to a template nucleic acid, such as an mRNA, and may or may not comprise one or more additional domains that may be considered a second (e.g., 5') domain that does not hybridize to the template nucleic acid, e.g., a non-template sequence domain as described in more detail below. The sequence of the template binding domain may be independently defined or arbitrary. In certain embodiments, the template binding domain has a defined sequence, e.g., poly dT or a gene specific sequence. In other embodiments, the template binding domain has an arbitrary sequence (e.g., a random sequence such as a random hexamer sequence). The length of the template binding domain may vary, but in some cases, the length of this domain ranges from 5 to 50 nt (e.g., 6 to 25 nt, e.g., 6 to 20 nt).

[0128] The single product nucleic acid primer is modified or otherwise non-naturally occurring. It may or may not contain one or more nucleotides (or analogs thereof). For example, the single product nucleic acid primer may contain one or more nucleotide analogs (e.g., LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, etc.), linkage modifications (e.g., phosphorothioates, 3'-3' reverse conjugates and 5'-5' reverse conjugates), 5' and / or 3' end modifications (e.g., 5' and / or 3' amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that confers a desired functionality to the single product nucleic acid primer.

[0129] In some cases, the single product nucleic acid primer may include a 5' adapter sequence (e.g., a defined nucleotide sequence 5' of the 3' hybridization domain of the single product nucleic acid primer), which may serve various purposes in downstream applications. In some cases, the 5' adapter sequence may function as a primer binding site for further amplification or, for example, nested amplification or suppression amplification.

[0130] In some cases, one or more of the primers or oligonucleotides used (including, for example, single product nucleic acid primers, template switch oligonucleotides, etc.) may contain two or more domains. For example, the primers or oligonucleotides may contain a first (e.g., 3') domain that hybridizes to the template and a second (e.g., 5') domain that does not hybridize to the template. The sequences of the first and second domains may be independently defined or arbitrary. In certain aspects, the first domain has a defined sequence and the sequence of the second domain is defined or arbitrary. In other aspects, the first domain has an arbitrary sequence (e.g., a random sequence such as a random hexamer sequence) and the sequence of the second domain is defined or arbitrary. In some cases, the sequences of both domains are defined. The primers used in the disclosed methods (including, for example, single product nucleic acid primers, template switch oligonucleotides, etc.) contain two or more domains, but one or more of the domains may contain a non-template sequence as described below.

[0131] Polymerase In some cases, the polymerase mixed into the template switching reverse transcription reaction mixture is capable of template switching, where the polymerase uses a first nucleic acid strand as a template for polymerization, and then switches to the 3' end of a second template nucleic acid strand to continue the same polymerization reaction. In some cases, the polymerase capable of template switching is a reverse transcriptase. Reverse transcriptases capable of template switching that are useful for carrying out the methods of the present disclosure include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, retroplasmid reverse transcriptase, retron reverse transcriptase, bacterial reverse transcriptase, group II intron-derived reverse transcriptase, and mutants, variants, derivatives, or functional fragments thereof, such as RNase H minus enzymes or RNase H reduced enzymes. For example, the reverse transcriptase can be Moloney murine leukemia virus reverse transcriptase (MMLV RT) or Bombyx mori reverse transcriptase (e.g., Bombyx moriR2 non-LTR element reverse transcriptase). Polymerases capable of template switching and useful for carrying out the methods of the present disclosure are commercially available and include SMARTScribe™ and PrimeScript™ reverse transcriptases available from Takara Bio USA, Inc. (Mountain View, Calif.).

[0132] The template switching reverse transcription reaction of the disclosed method comprises a terminal transferase The present invention may include the use of a polymerase having terminal transferase activity. For example, the polymerase (e.g., a reverse transcriptase such as MMLV RT) combined with the reaction mixture has terminal transferase activity such that it can add a homonucleotide region (e.g., a homotrinucleotide such as CCC) to the 3' end of the nascent strand, and the 3' hybridization domain of the template switch oligonucleotide comprises a homonucleotide region (e.g., a homotrinucleotide such as GGG) that is complementary to the region at the 3' end of the nascent strand. In another embodiment, when the polymerase with terminal transferase activity adds a nucleotide region (e.g., a trinucleotide region) to the 3' end of the nascent strand, the 3' hybridization domain of the template switch oligonucleotide comprises a heterotrinucleotide (e.g., a r(C / G)3 oligonucleotide) that comprises a nucleotide containing a cytosine and a nucleotide containing a guanine, and the heterotrinucleotide region of the template switch oligonucleotide is complementary to the 3' end of the nascent strand. Examples of 3' hybridization domains and template switch oligonucleotides are further described in U.S. Patent No. 5,962,272, the disclosure of which is incorporated herein by reference.

[0133] A polymerase with terminal transferase activity can catalyze the addition of deoxyribonucleotides to the 3' hydroxyl end of an RNA or DNA molecule. In certain embodiments, when a polymerase reaches the 5' end of a template, the polymerase can introduce one or more additional nucleotides not encoded by the template at the 3' end of the nascent strand. For example, when a polymerase has terminal transferase activity, the polymerase may be able to introduce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional nucleotides at the 3' end of the nascent strand. The nucleotides may all be the same (e.g., creating a homonucleotide region at the 3' end of the nascent strand), or one or more of the nucleotides may be different from the other nucleotide(s) (e.g., creating a heteronucleotide region at the 3' end of the nascent strand). In certain aspects, the terminal transferase activity of the polymerase adds a homonucleotide region of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more identical nucleotides (e.g., all dCTP, all dGTP, all dATP, or all dTTP). For example, according to one embodiment, the polymerase is MMLV reverse transcriptase (MMLV RT). MMLV RT introduces additional nucleotides (mostly dCTP, e.g., three dCTPs) at the 3' end of the nascent strand. As described in more detail elsewhere herein, these additional nucleotides may be useful for hybridizing the 3' hybridization domain of the template switch oligonucleotide to the 3' end of the nascent strand, for example, to facilitate template switching of the polymerase from the template to the template switch oligonucleotide.

[0134] The reverse transcriptase used in the disclosed method may in some cases be a thermosensitive polymerase, i.e., a polymerase that is not thermostable. Such thermosensitive polymerases may become inactive at temperatures above their active temperature range. For example, in some cases, thermosensitive polymerases may become inactive or have significantly reduced activity after exposure to temperatures of 75° or higher, 80° or higher, 85° or higher, 90° or higher, or 95° or higher.

[0135] When a reverse transcriptase is used, the reverse transcriptase may be combined with the reaction mixture at a final concentration sufficient to generate the desired amount of RT reaction product, e.g., the desired amount of single product nucleic acid. In certain embodiments, the reverse transcriptase (e.g., MMLV RT, Bombyx mori RT, etc.) may be combined with the reaction mixture at a concentration of 0.1 to 200 units per μL (U / μL) (0.5 to 100 U / μL, e.g., 1 to 50 U / μL, etc. (5 to 25 U / μL, e.g., 20 U / μL). L) at a final concentration of 0.1 mM NaCl.

[0136] Non-templated and non-template sequences The described method aspects may, in some cases, include the use of non-template sequences. The terms "non-templated sequence" and "non-template sequence" generally refer to sequences involved in the disclosed methods that do not correspond to a template (e.g., sequences that are not present in the template, do not have a complementary sequence in the template, or are unlikely to be present in the template or have a complementary sequence in the template). A non-template sequence is a sequence that is not templated by a template, e.g., an RNA or DNA template, and thus can be added in the absence of a corresponding template, e.g., during an extension reaction, e.g., nucleotides added by a polymerase with terminal transferase activity that is not template-directed. Addition of a non-template sequence to a nucleic acid need not necessarily be limited to an extension reaction. For example, in some cases, a non-template sequence may be added through ligation of the non-template sequence to a nucleic acid. In some cases, a non-template sequence may be added through a transposase-mediated reaction, e.g., a tagmentation reaction that adds the non-template sequence to the nucleic acid of interest. Thus, the non-template sequences can be diverse and can be added to the template sequence through a variety of means.

[0137] Non-template and non-templated sequences may refer, but are not limited to, sequences present in a primer, template switch oligonucleotide, or transposon that do not hybridize to a nucleic acid template (such sequences may in some cases be referred to as non-hybridizing sequences). Non-template sequences can vary in both size and composition. In some cases, the non-template sequence, e.g., a non-template sequence present in a template switch oligonucleotide or primer, may be in the range of 10 nt to 1000 nt or more, for example, but not limited to, 10 nt to 900 nt, 10 nt to 800 nt, 10 nt to 700 nt, 10 nt to 600 nt, 10 nt to 500 nt, 10 nt to 400 nt, 10 nt to 300 nt, 10 nt to 200 nt, 10 nt to 100 nt, 10 nt to 90 nt, 10 nt to 80 nt, 10 nt to 70 nt, 10 nt to 60 nt, 10 nt to 50 nt, 10 nt to 40 nt, 10 nt to 30 nt, and 10 nt to 20 nt.

[0138] In some cases, the non-template sequence may be included in the 3' hybridization domain of the template switch oligonucleotide, as described above. The non-template sequence, when present in the 3' hybridization domain of the template switch oligonucleotide, may comprise or consist of a heteropolynucleotide, which may vary in length from 2 nt to 10 nt, such as 3-7 nt in length, inclusive. In some cases, the non-template sequence present in the 3' hybridization domain of the template switch oligonucleotide may comprise or consist of a homopolynucleotide, which may vary in length from 2 nt to 10 nt, such as 3-7 nt in length, inclusive.

[0139] The non-template sequence present in the oligonucleotide or primer may be present at the 5' end of the oligonucleotide or primer, in which case the sequence may be referred to as the 5' non-template sequence. In some cases, only one oligonucleotide or primer may contain a non-template sequence (e.g., a 5' non-template sequence). In some cases, two or more oligonucleotides and / or primers used in the reactions of the present disclosure may contain a non-template sequence (e.g., a 5' non-template sequence). When two or more oligonucleotides and / or primers contain a non-template sequence, different non-template sequences may be used. In some cases, when two or more oligonucleotides and / or primers have a 5' non-template sequence, such sequences may have the same 5' non-template sequence.

[0140] In some cases, the non-template sequence (including, for example, the 5' non-template sequence) may include one or more restriction endonuclease recognition sites. In some cases, one or more restriction endonuclease recognition sites may be introduced into a nucleic acid of interest to render the resulting nucleic acid operable, for example, by cleaving the nucleic acid of interest at one or more of the introduced restriction endonuclease recognition sites.

[0141] In some cases, the non-template sequence (e.g., including the 5' non-template sequence) may include one or more primer binding sites. In some cases, one or more primer binding sites may be introduced into a nucleic acid of interest to render the resulting nucleic acid further amplifiable (e.g., including amplifying all or a portion of the nucleic acid using one or more primer binding sites).

[0142] Useful primer binding sites vary widely depending on the desired complexity of the primer binding site and the corresponding primer. In some cases, useful primer binding sites include primer binding sites that are complementary to II A primers (e.g., as available from Takara Bio USA, Inc. (Mountain View, CA)). According to one embodiment, the oligonucleotide or primer used in generating the product double-stranded cDNA comprises a non-template sequence that includes a II A primer binding site. According to one embodiment, the nucleic acid used in the end capture reaction comprises a non-template sequence that includes a II A primer binding site.

[0143] In some cases, the non-template sequences (e.g., including 5' non-template sequences) may comprise one or more barcode sequences, and in some cases, such barcode sequences may be or may include unique molecular identifier (UMI) domains and / or barcoded unique molecular identifier (BUMI) domains. UMI and BUMI nucleic acids and their use in various applications are further described in U.S. Patent Application Publication No. US20150072344, the disclosure of which is incorporated herein by reference in its entirety.

[0144] In some cases, for example, after a sequencing reaction in which the barcodes are sequenced, one or more barcode sequences of the non-template sequence may trace back to the source of the generated nucleic acid. For example, in some cases, a non-template sequence is introduced during the reaction that includes a barcode specific to the source of the template (e.g., a sample, a well, a cell, etc.). Such source-identifying barcodes may be referred to herein as "source barcode sequences," and such sequences may vary and may be assigned a term based on the source that the barcode identifies. Source barcodes may include, for example, a sample barcode sequence that traces back to the sample from which the sequenced nucleic acid was generated, a well barcode sequence that traces back to the well (e.g., a well of a multi-well plate) from which the sequenced nucleic acid was generated, a droplet barcode sequence that traces back to the droplet from which the sequenced nucleic acid was generated, a cell barcode sequence that traces back to the cell (e.g., a cell of a multicellular sample) from which the sequenced nucleic acid was generated, and a marker barcode sequence that traces back to the sample from which the sequenced nucleic acid was generated. Barcodes may include coding sequences, etc. Barcodes can be used in a variety of procedures, including, for example, when pooling nucleic acids after barcoding, e.g., prior to sequencing.

[0145] In some cases, for example, the non-template sequence present in the oligonucleotide and / or nucleic acid primer comprises a sequencing platform adaptor construct. By "sequencing platform adapter construct" is meant a nucleic acid construct that includes at least a portion of a nucleic acid domain (e.g., a sequencing platform adapter nucleic acid sequence) or its complement for use with an applicable sequencing platform (such as a sequencing platform supplied by Illumina® (e.g., the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), a sequencing platform supplied by Ion Torrent™ (e.g., the Ion PGM™ and / or Ion Proton™ sequencing systems), a sequencing platform supplied by Pacific Biosciences (e.g., the PACBIO RS II sequencing system), a sequencing platform supplied by Life Technologies™ (e.g., the SOLiD sequencing system), a sequencing platform supplied by Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or any other applicable sequencing platform).

[0146] In certain embodiments, the non-template sequence comprises a sequencing platform adaptor construct that includes a nucleic acid domain that is a domain (e.g., a "capture site" or "capture sequence") that specifically binds to a surface-attached sequencing platform oligonucleotide (e.g., a P5 or P7 oligonucleotide attached to the surface of a flow cell of an Illumina® sequencing system) and a sequencing primer binding domain (e.g., a domain to which a Read1 or Read2 primer of an Illumina® platform can bind). The sequencing platform adaptor construct may include a nucleic acid domain (e.g., a "sequencing adaptor") of any length and sequence suitable for the sequencing platform in question. In certain embodiments, the nucleic acid domain may be 4-200 nt in length. For example, the nucleic acid domain may be 4-100 nt in length (e.g., 6-75 nt, 8-50 nt, or 10-40 nt in length, etc.). According to certain embodiments, the sequencing platform adaptor construct comprises a nucleic acid domain that is 2-8 nt in length (e.g., 9-15 nt, 16-22 nt, 23-29 nt, or 30-36 nt in length).

[0147] The nucleic acid domain may have a length and sequence that allows a polynucleotide (e.g., an oligonucleotide) used in a corresponding sequencing platform to specifically bind to the nucleic acid domain, for example, during solid-phase amplification and / or sequencing by synthesis of a cDNA insert adjacent to the nucleic acid domain. Examples of nucleic acid domains include the P5 (5'-AATGATACGGCGACCACCGA-3') (SEQ ID NO: 17), P7 (5'-CAAGCAGAAGACGGCATACGAGAT-3') (SEQ ID NO: 18), Read1 primer (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') (SEQ ID NO: 19), and Read2 primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3') (SEQ ID NO: 20) domains used in Illumina®-based sequencing platforms. Another example of a nucleic acid domain is the A adaptor (5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3') used in Ion Torrent™-based sequencing platforms. SEQ ID NO:21) and the P1 adaptor (5'-CCTCTCTATGGGCAGTCGGTGAT-3') (SEQ ID NO:22) domain.

[0148] The nucleotide sequence of the non-template sequence domain useful for sequencing on a given sequencing platform may vary and / or may change over time. The adaptor sequence is typically provided by the manufacturer of the sequencing platform (e.g., in the technical documentation provided with the sequencing system and / or available on the manufacturer's website). Based on such information, the sequence of the sequencing platform adaptor construct of the non-template sequence (e.g., template switch oligonucleotide and / or single product nucleic acid primer and / or the like) may be designed to include all or part of one or more nucleic acid domains in a configuration that allows sequencing of the nucleic acid insert (the insert corresponding to the template nucleic acid) on the given platform. Sequencing platform adaptor constructs that may be included in the non-template sequence and other nucleic acid reagents described herein are further described in U.S. Patent Application Serial No. 14 / 478,978, published as US2015-0111789A1, the disclosure of which is incorporated herein by reference.

[0149] Non-template sequences may be added to the relevant nucleic acid, such as an oligonucleotide, a nucleic acid primer, a prepared dsDNA, etc., by various means. For example, as described above, non-template sequences may be added through the action of a polymerase with terminal transferase activity. For example, non-template sequences present in a primer or oligonucleotide may be introduced into the product nucleic acid during an amplification reaction. In some cases, non-template nucleic acid sequences may be directly attached to a nucleic acid (e.g., to a primer or oligonucleotide prior to amplification, to a nucleic acid amplification product, etc.). There are various methods for directly attaching non-template sequences to a nucleic acid, including, but not limited to, ligation, chemical synthesis / ligation, enzymatic addition of nucleotides (e.g., addition by a polymerase with terminal transferase activity), etc.

[0150] In some cases, the disclosed methods may include attaching a sequencing platform adapter construct to the end of the nucleic acid. For example, in some cases, the oligonucleotides and / or primers used in the disclosed methods may not include a sequencing platform adapter construct, i.e., the desired sequencing platform adapter construct may be attached after the nucleic acid of interest is generated. The adapter construct attached to the end of the nucleic acid of interest or a derivative thereof may include any sequence element useful in downstream sequencing applications, including any of the elements described above with respect to any sequencing platform adapter construct of the oligonucleotides and / or primers of the methods described herein. For example, the adapter construct attached to the end of the nucleic acid of interest or a derivative thereof may include a nucleic acid domain, or a complement thereof, selected from the group consisting of a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof.

[0151] Attachment of the sequencing platform adaptor constructs may be performed using any suitable approach. In certain embodiments, the adaptor constructs are attached to the ends of the product nucleic acid or derivatives thereof using the same or similar approach as the "seamless" cloning method. The seamless method includes steps such as restriction enzyme analysis and digestion, DNA end repair, dephosphorylation, ligation, enzyme inactivation and cleanup, and pairing of nucleic acid material. The seamless ligation method eliminates one or more rounds of loss of corresponding sequences. Examples of seamless ligation methods include the In-Fusion® cloning system available from Takara Bio USA, Inc. (Mountain View, CA), SLIC (Sequence and Ligase Independent Cloning) as described in Li & Elledge (2007) Nature Methods 4:251-256, Gibson assembly as described in Gibson et al. (2009) Nature Methods 6:343-345, CPEC (Circular Polymerase Extension Cloning) as described in Quan & Tian (2009) PLoS ONE 4(7):e6441, SLiCE (Seamless Ligation Cloning Extract Method) as described in Zhang et al. (2012) Nucleic Acids Research 40(8):e55, and GeneArt® seamless cloning technology by Life Technologies (Carlsbad, CA).

[0152] Any suitable approach may be used to provide additional nucleic acid sequencing domains to the nucleic acid of interest or its derivatives that have only a portion of the sequencing domains useful or necessary for the sequencing platform of interest.For example, a PCR primer with an adapter sequence at its 5' end (e.g., 5' of the primer region that is complementary to the nucleic acid of interest or its derivatives) may be used to amplify the nucleic acid of interest or its derivatives, so that the amplicon contains the adapter sequence in the original product nucleic acid and the adapter sequence in the primer in any desired configuration.Other approaches may also be used, including approaches based on seamless cloning methods, restriction digestion / ligation, etc.

[0153] Additional method parameters As outlined above, the methods described herein may include certain nucleic acid reactions, including, for example, template switching reverse transcription reactions, nucleic acid amplification reactions, end capture reactions, tagmentation reactions, and the like. The reaction mixture components in such reactions are combined under conditions sufficient to generate a product of the reaction. For example, in some cases, reaction components of a template switching reverse transcription reaction are combined under conditions sufficient to generate a product double-stranded cDNA. In some cases, reaction components of a nucleic acid amplification reaction are combined under conditions sufficient to generate an amplified product nucleic acid. In some cases, reaction components of an end capture reaction are combined under conditions sufficient to generate an end captured nucleic acid. In some cases, reaction components of a tagmentation reaction are combined under conditions sufficient to generate a tagmented nucleic acid.

[0154] "Conditions sufficient to produce" nucleic acids of the present disclosure refer to reaction conditions that allow the relevant nucleic acids and / or other reaction components in the reaction to interact with each other in a desired manner. For example, in some cases, the conditions may be sufficient for the nucleic acids of the reaction mixture to hybridize. In some cases, the conditions may be sufficient for the enzymes of the reaction mixture to catalyze a chemical process (e.g., polymerization, hydrolysis, etc.). Providing suitable reaction conditions may include selecting reaction mixture components, their concentrations, and reaction temperatures to create an environment in which the relevant process proceeds (e.g., an environment in which the relevant nucleic acids hybridize with each other in a sequence-specific manner, an environment in which the relevant polymerase polymerizes to extend the nucleic acid, etc.). In addition to the specific nucleic acids of the reaction (e.g., template nucleic acid, oligonucleotides, primers, etc.), the reaction mixture may include buffer components to establish an appropriate pH, salt concentration (e.g., KCl concentration), etc. Conditions sufficient to produce double-stranded nucleic acid complexes may include conditions suitable for hybridization (also referred to as "hybridization conditions").

[0155] To achieve favorable reaction conditions, the reaction mixture components, their concentrations and reaction temperature are selected to obtain one Suitable reaction conditions may include creating an environment in which the polymerases described above are active and / or the associated nucleic acids in the reaction can interact (e.g., hybridize) with one another in a desired manner. In addition to the reaction components, the reaction mixture contains the appropriate pH, salt concentration (e.g., KCl concentration), metal cofactor concentration (e.g., Mg, Zn, Fe, Fe+ ... 2+ or Mn 2+ The reaction mixture may include buffer components that establish the concentration, etc. Other components may be included, such as one or more nuclease inhibitors (e.g., RNase inhibitors and / or DNase inhibitors), one or more additives that promote amplification / replication of GC-rich sequences (e.g., GC-Melt™ Reagent (Takara Bio USA, Inc. (Mountain View, Calif.)), betaine, DMSO, ethylene glycol, 1,2-propanediol, or combinations thereof), one or more molecular crowding agents (e.g., polyethylene glycol, etc.), one or more enzyme stabilizing components (e.g., DTT present at a final concentration in the range of 1-10 mM (e.g., 5 mM)), and / or any other reaction mixture components useful for promoting polymerase-mediated extension reactions and / or template switching.

[0156] The one or more reaction mixtures may be at a suitable pH for primer extension reactions and / or template switching. In certain embodiments, the pH of the reaction mixture is in the range of 5 to 9 (such as 7 to 9, including 8 to 9, e.g., 8 to 8.5). In some cases, the reaction mixture comprises a pH adjuster. Suitable pH adjusters include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphate buffer solution, citrate buffer solution, and the like. For example, the pH of the reaction mixture can be adjusted to a desired range by adding an appropriate amount of the pH adjuster.

[0157] Suitable temperature ranges for primer extension reactions can vary depending on factors such as the particular polymerase used, the melting temperature of any primers used, etc. In some cases, a reverse transcriptase (e.g., MMLV reverse transcriptase) may be used, and reaction mixture conditions sufficient for reverse transcriptase-mediated extension of hybridized primers include subjecting the reaction mixture to a temperature in the range of 4°C to 72°C (16°C to 70°C, e.g., 37°C to 50°C, 40°C to 45°C, etc., inclusive of 42°C).

[0158] In some cases, the methods described herein may include denaturing the template, for example, by exposing a reaction mixture containing the template to a temperature sufficient to denature secondary structures of the template. Depending on the circumstances, denaturation may occur before or after one or more reaction components are added to the reaction mixture, and in some cases, before the initiation of transcription, e.g., reverse transcription, to generate a single product nucleic acid. Useful denaturation temperatures can range from less than 50°C to more than 100°C, including, but not limited to, 50°C or higher, 55°C or higher, 65°C or higher, 70°C or higher, 72°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, etc.

[0159] In some cases, the methods provided may include isolating and / or purifying the final nucleic acid product (e.g., a nucleic acid library) and / or intermediate nucleic acid products (e.g., double-stranded product cDNA). Any convenient purification method may be used, including, but not limited to, nucleic acid precipitation (i.e., alcohol precipitation), gel purification, and the like.

[0160] In some cases, the methods provided may include using an amplification polymerase, for example, for use in amplifying the generated double stranded cDNA, the generated nucleic acid library, etc. Any convenient amplification polymerase may be used, including, but not limited to, DNA polymerases, including thermostable polymerases. Useful amplification polymerases include, for example, Taq DNA polymerase, Pfu DNA polymerase, derivatives thereof, etc. In some cases, the amplification polymerase may be a hot start polymerase. It may be a polymerase, for example, but not limited to, hot-start Taq DNA polymerase, hot-start Pfu DNA polymerase, and the like.

[0161] The amplification polymerase may be combined with the reaction mixture such that the final concentration of the amplification polymerase is sufficient to generate a desired amount of product nucleic acid, e.g., a desired amount of amplified product double-stranded cDNA, a desired amount of library nucleic acid, etc. In certain embodiments, the amplification polymerase (e.g., a thermostable DNA polymerase, a hot-start DNA polymerase, etc.) is present in the reaction mixture at a final concentration of 0.1-200 units per μL (U / μL) (0.5-100 U / μL, such as 1-50 U / μL, including 5-25 U / μL, e.g., 20 U / μL).

[0162] The nucleic acid reaction, e.g., amplification reaction, of the disclosed methods may include combining dNTPs into the reaction mixture. In certain embodiments, each of the four natural dNTPs (dATP, dGTP, dCTP, and dTTP) is added to the reaction mixture. For example, dATP, dGTP, dCTP, and dTTP may be added to the reaction mixture such that the final concentration of each dNTP is 0.01-100 mM, such as 0.1-10 mM, including 0.5-5 mM, (e.g., 1 mM). In some cases, one or more nucleotides added to the reaction mixture may be unnatural nucleotides, e.g., modified nucleotides having a linking moiety or other moiety (e.g., a fluorescent moiety) attached to the nucleotide, a nucleotide analog, or any other type of unnatural nucleotide useful in the disclosed methods or applicable downstream applications.

[0163] The reaction mixture may be exposed to various temperatures to drive various aspects of the reaction, including, but not limited to, nucleic acid denaturation / melting, nucleic acid hybridization / annealing, polymerase-mediated stretching / extension, etc. The temperatures at which various processes are performed may be referred to according to the process performed, including, for example, melting temperature, annealing temperature, stretching temperature, etc. The optimal temperatures for such processes will vary depending, for example, on the polymerase used, the characteristics of the nucleic acid, etc. The optimal temperatures for a particular polymerase, including reverse transcriptase and amplification polymerase, can be readily obtained from reference texts. Optimal temperatures for nucleic acids, such as annealing and melting temperatures, can be readily calculated based on known characteristics of the nucleic acid in question, including, for example, total length, hybridization length, G / C content, predicted secondary structure, etc.

[0164] According to certain embodiments, the methods of the present disclosure may include isolating, amplifying and / or analyzing (e.g., sequencing) deoxyribonucleic acid (DNA). When the methods of the present disclosure include isolating, amplifying and / or analyzing DNA, the DNA used may be referred to as a DNA template (or, in some cases, as template DNA). The template DNA may be any type of DNA (or subtype thereof), including, but not limited to, genomic DNA (e.g., animal genomic DNA (e.g., mammalian genomic DNA (e.g., human genomic DNA, rodent genomic DNA (e.g., mouse, rat, etc.)), mitochondrial DNA, or any combination of these types of DNA or subtypes thereof).

[0165] In certain embodiments, genomic DNA (gDNA) may be isolated and / or processed for analysis, as desired. For example, in some cases, the methods provided may include preparing one or more libraries from a sample containing RNA, and may further include isolating, processing, and / or analyzing gDNA from the sample. Thus, in some cases, a sample may include a sample containing both RNA and DNA (e.g., gDNA), including, for example, a nucleic acid sample isolated from multiple cells, a sample isolated from a single cell. For example, in some cases, the methods disclosed herein may include isolating RNA, DNA, and / or analyzing gDNA from a single cell. The method may include isolating, processing and / or analyzing the RNA, for example where processing the RNA includes preparing two or more libraries (e.g., an expression library and an immune cell receptor repertoire library) from the RNA sample.

[0166] Isolation, processing and / or analysis of gDNA may be performed for a variety of purposes. For example, in some cases, gDNA from a sample may be sequenced to obtain genomic sequence information. Such sequencing of gDNA from a sample of interest may, in some cases, include sequencing an immune locus or more than one immune locus. "Immune locus" generally refers to the locus of any immune-related gene, including genes associated with immune system processes (such as those identified by Gene Ontology (GO) Accession Number GO:0002376 (available online at geneontology.org)), including, for example, B cell-mediated immunity, B cell selection, T cell-mediated immunity, T cell selection, activation of immune responses, antigen processing and presentation, antigen sampling in mucosa-associated lymphoid tissue, basophil-mediated immunity, eosinophil-mediated immunity, hematopoietic differentiation, hematopoietic proliferation, immune efflux ... These include, but are not limited to, genes associated with vector processes, immune responses, induction of the immune system, immune memory processes, leukocyte activation, leukocyte homeostasis, leukocyte-mediated immunity, leukocyte migration, lymphocyte costimulation, lymphocyte-mediated immunity, mast cell-mediated immunity, myeloid cell homeostasis, myeloid leukocyte-mediated immunity, natural killer cell-mediated immunity, regulation of negative immune system processes, neutrophil-mediated immunity, regulation of positive immune system processes, production of mediator molecules for immune responses, regulation of immune system processes, diversification of immune receptors at the somatic cell level, and tolerance induction.

[0167] In some cases, the immune locus that may be sequenced and / or otherwise analyzed in the methods of the present disclosure may be a TCR locus. In some cases, the immune locus that may be sequenced and / or otherwise analyzed in the methods of the present disclosure may be a BCR locus. In some cases, sequencing of gDNA of the immune locus may allow for coordinated analysis with NGS analysis of one or more libraries generated herein, including, for example, expression libraries and / or immune cell receptor repertoire libraries. In some cases, gDNA analysis performed in the methods provided may include whole genome sequencing.

[0168] Compositions and kits Aspects of the present disclosure also include compositions and kits. The compositions and kits of the present disclosure may include, for example, one or more of any of the reaction mixture components described above with respect to the methods of the present disclosure. For example, the compositions and kits of the present disclosure may include a nucleic acid sample (e.g., an RNA sample, a sample in which RNA and DNA are combined, etc.), an amplification polymerase (e.g., a thermostable polymerase, etc.), a reverse transcriptase (e.g., a reverse transcriptase capable of template switching, etc.), a template switch oligonucleotide, an end capture primer, an immune receptor specific primer, one or more components of a tagmentation reaction, dNTPs, salts, metal cofactors, one or more nuclease inhibitors (e.g., RNase inhibitors and / or DNase inhibitors), one or more molecular crowding agents (e.g., polyethylene glycol, etc.), one or more enzyme stabilizing components (e.g., DTT), or any other desired kit component(s).

[0169] In some cases, the components of the compositions and / or kits of the present disclosure may be provided as a "cocktail," which as used herein refers to a collection or combination of two or more different but similar components in one container. Cocktails useful in the kits of the present disclosure include, but are not limited to, "primer cocktails," which may vary in composition and may include, for example, a cocktail of two or more primers (e.g., including a terminal amplification primer and an immune receptor-specific primer). Cocktails useful in the kits of the present disclosure include, but are not limited to, "primer cocktails," which may vary in composition and may include, for example, a cocktail of two or more primers (e.g., including a terminal amplification primer and an immune receptor-specific primer). Examples of such cocktails include, but are not limited to, a "tagmentation cocktail." Such cocktails can vary in composition and can include, for example, a cocktail of two or more components of a tagmentation reaction (e.g., including a transposon and a transposase).

[0170] In certain embodiments, the kits of the present disclosure include reagents for isolating nucleic acids from a nucleic acid source of interest. The reagents may be suitable for isolating nucleic acid samples from a variety of DNA or RNA sources, including single cells, cultured cells, tissues, organs, or organisms. The kits of the present disclosure may include reagents for isolating nucleic acid samples from fixed cells, tissues, or organs, such as formalin-fixed, paraffin-embedded (FFPE) tissues. Such kits may include one or more deparaffinizing agents, one or more agents suitable for decrosslinking nucleic acids, and / or the like.

[0171] The components of the kit of the present disclosure may be in separate containers, or multiple components may be in one container. For example, components for performing the template switching reaction and components for preparing the library (e.g., components used after splitting the generated double-stranded cDNA) may be in different tubes. In some cases, the template switch oligonucleotide and the first strand cDNA primer may be in the same tube or in different tubes. In some cases, one or more immune receptor-specific primers and one or more terminal (e.g., 5'-end) primers may be in the same tube or in different tubes. In some cases, one or more terminal (e.g., 5'-end or 3'-end) primers and one or more post-tagmentation primers may be in the same tube or in different tubes. In some cases, deoxyribonucleotide triphosphates (dNTPs) may be in the same tube as the reverse transcriptase, the amplification polymerase, or one or more primers or oligonucleotides.

[0172] In certain embodiments, the kits provided may include any combination of a 5' amplification primer, an immune cell receptor-specific amplification primer (i.e., an immune receptor-specific primer), and a terminal amplification primer. For example, the kit may include a 5' amplification primer that binds to a primer binding site added by the template switch oligonucleotide, in combination with an immune cell receptor-specific amplification primer that specifically binds to a region (e.g., a constant region) of one or more immune cell receptor polypeptides (e.g., a TCR alpha chain, a TCR beta chain, an immunoglobulin chain), and a terminal amplification primer used in terminal capture (e.g., a terminal amplification primer that binds to a primer binding site added in a terminal capture process (e.g., a tagmentation reaction, etc.)).

[0173] In certain cases, the provided kits may include one or more components for performing a template switching reverse transcription reaction. Such components include, but are not limited to, those described herein, including, for example, template switching oligonucleotides, primers, reverse transcriptases, etc. Such components, e.g., oligonucleotides and primers, may in some cases include adapter sequences. For example, in some cases, the provided template switching oligonucleotides may include a 5' adapter sequence.

[0174] In certain cases, the kits provided may include one or more components for performing a tagmentation reaction. For example, such kits may include one or more of the following reagents: a transposon nucleic acid including a post-tagmentation amplification primer binding domain, a post-tagmentation amplification primer that hybridizes to the post-tagmentation amplification primer binding domain, a transposase (e.g., Tn5 transposase), or a combination of any of these. The composition may include any of a number of combinations thereof, or any combination of several others (which may include one or more of the additional ingredients described herein), or any combination thereof.

[0175] In certain cases, the kits provided may be used in conjunction with automated systems (e.g., Takara Bio The kit may include one or more components for performing multiple reactions on a 350-well plate (i.e., an array chip) manufactured by Eppendorf, USA. The provided kit may include a multiwell plate (i.e., an array chip). The multiwell array chip may include the template switch oligonucleotides of the present disclosure and / or any other primers in the wells of the multiwell array chip (e.g., in dry form).

[0176] In addition to the components described above, the kits of the present disclosure may further include instructions for using the components of the kit to perform, for example, the methods of the present disclosure as described above. Additionally, for example, if the primers and / or oligonucleotides of the kit include a BUMI domain, the kit may further include programming for analyzing the results, including, for example, decoding the coded BUMI domain, counting unique molecular species, and the like. The instructions and / or analytical programming are generally recorded on a suitable recording medium. The instructions and / or programming may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, a label on the container of the kit or its components (i.e., a label associated with the package or subpackage), and the like. In another embodiment, the instructions may be present as an electronic storage data file on a suitable computer readable storage medium, for example, a CD-ROM, a diskette, a hard disk drive (HDD), and the like. In yet another embodiment, the actual instructions are not included in the kit, but a means is provided for obtaining the instructions from a remote source, for example, via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. With respect to the instructions, this means of obtaining the instructions is recorded on a suitable substrate.

[0177] The composition of the present disclosure may be present in any suitable environment. According to one embodiment, the composition of the present disclosure is contained in a reaction tube (e.g., 0.2 mL tube, 0.6 mL tube, 1.5 mL tube, etc.), a well, a microfluidic chamber, a droplet, or other suitable container. In certain aspects, the composition of the present disclosure is contained in two or more (e.g., a plurality) reaction tubes or wells (e.g., a plate such as a 96-well plate, a multi-well plate (e.g., containing about 1000, 5000, or 10,000 or more wells). The tube and / or plate may be made of any suitable material, such as polypropylene, PDMS, or aluminum. The container may be treated to reduce adsorption of nucleic acids to the container walls. In certain aspects, the tube and / or plate containing the composition of the present disclosure is capable of efficiently transferring heat to the composition of the present disclosure (e.g., when placed in a heat block, water bath, thermocycler, and / or the like) to allow the temperature of the composition to be changed over a short period of time, such as required to cause a particular enzymatic reaction to occur. According to certain embodiments, the compositions of the present disclosure are contained in thin-walled polypropylene tubes, or thin-walled polypropylene wells or plates with a material with high thermal conductance (such as aluminum). In some cases, the compositions of the present disclosure may be contained in droplets. In certain embodiments, it is convenient to perform the reaction on a solid surface or bead, in which case the single product nucleic acid primer and / or template switch oligonucleotide, or one or more other primers, may be attached to a solid support or bead by methods known in the art (such as biotin binding or covalent binding) and the reaction may proceed on the support. Alternatively, oligos may be synthesized directly on a solid support, for example as described in Macosko, EZ et.al, Cell 161, 1202-1214, May 21, 2015.

[0178] Other suitable environments for the compositions of the present disclosure include, for example, microfluidic chips (e.g., "lab-on-a-chip devices", e.g., microfluidic devices with channels and inlets). The compositions of the present disclosure may be placed in an instrument configured to bring the composition to a desired temperature, e.g., a temperature-controlled water bath, a heat block, a heat block adapter, etc. The instrument configured to bring the composition to a desired temperature may be configured to bring the composition to a series of different desired temperatures, each for a suitable period of time (e.g., the instrument may be a thermocycler).

[0179] usefulness The methods of the disclosure are useful in a variety of applications, including immune cell receptor repertoire analysis and / or applications employing analysis of two or more separately prepared sequencing libraries, including where such analyses can be performed sequentially or simultaneously.

[0180] As mentioned above, in some cases, data from two libraries prepared separately may be coordinated. Such coordinated data may serve a common or related purpose. For example, in some cases, data from an immune cell receptor repertoire library may be coordinated with data from an expression library. In some embodiments, sequencing of an immune cell receptor repertoire library may provide information regarding the identity of the repertoire of immune receptors expressed in a cell, a cell population, or a subject or subject population, and differential expression analysis of an expression library may provide information regarding relative gene expression levels, which may be coordinated with the immune receptor repertoire information. For example, in some cases, an expression library may provide differential expression information regarding the entire transcriptome of one or more cells (e.g., as part of a whole transcriptome analysis (WTA)), and such transcriptome information may be compared or coordinated with the immune receptor repertoire information of the same one or more cells. As another example, in some cases, an expression library can provide differential expression information regarding the expression of one or more immune-related genes (e.g., cytokines, interleukins, interleukin receptors, CD4, CD8, CD3, PD-1, etc.) in one or more cells, which expression information for such immune-related genes can be compared or coordinated with information about the immune receptor repertoire of the same one or more cells. Through such a process, relationships between the immune receptor repertoire of a cell or cell population and the expression of other genes (including immune and non-immune related genes) can be identified and / or further investigated.

[0181] Furthermore, the use of single product double stranded cDNA in parallel library preparations may allow for improved consistency of results and the ability to correlate results across data sets. Additionally, the use of single product double stranded cDNA in multiple library preparation methods is further useful in methods where reduced user input and "hands on time" is desired to generate two or more libraries, with or without subsequent coordination of data from the two or more libraries.

[0182] Applications of the disclosed methods include medical and research applications in which identification and / or screening of immune molecules is desirable, including human clinical research applications and preclinical research applications (e.g., preclinical research applications conducted in animal models such as rodents, small mammals, large mammals, non-human primates, etc.).

[0183] Libraries prepared by the methods disclosed herein and ready for sequencing can be analyzed using any convenient sequencing platform (e.g., The adapter sequences allow the library members to be sequenced using a sequencing platform such as Illumina®'s HiSeq™, MiSeq™ and Genome Analyzer™ sequencing systems, Ion Torrent™'s Ion PGM™ and Ion Proton™ sequencing systems, Pacific Biosciences' PACBIO RS II sequencing system, Life Technologies™'s SOLiD sequencing system, Roche's 454 GS FLX+ and GS Junior sequencing systems, or any other convenient sequencing platform. The provided methods are useful for generating one or more sequencing-ready libraries corresponding to a single sample of RNA starting material of interest, e.g., either mRNA, non-polyadenylated RNA (e.g., microRNA). In addition, the disclosed methods may be used to generate sequencing-ready cDNA libraries, either singly or in parallel, from a single RNA sample derived from an individual single cell, or from a related cell population.

[0184] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0185] Example 1: Combined gene expression analysis and TCR profiling from single cells An overall schematic showing the overall process used to generate libraries for use in combined single-cell derived differential gene expression analysis and TCR profiling is shown in FIG. 5.

[0186] Reverse transcription (RT) reaction and pre-amplification PCR The first part of the protocol involved generating double-stranded cDNA from input single-cell mRNA samples through a template-switching reverse transcription reaction. Single-cell mRNA samples were distributed into wells of a 96-well plate, and in each well, a T cell receptor (TCR) dT primer and reverse transcriptase (RT) were used to synthesize the first strand from the single-cell mRNA sample. The terminal transferase activity of RT tailed the synthesized first strand. The tailed nucleotides were hybridized to SMART-Seq Indexed Oligonucleotides to allow template switching of RT and extension of the synthesized first strand, including the index and pre-amplification PCR primer binding site sequences with the SMART-Seq Indexed Oligonucleotide as the template.

[0187] The products of the template switching reaction were then amplified using TCR dT Primer and Pre-Amp PCR Primer (hybridizing to the pre-amplification PCR primer binding site) to finally generate product double-stranded cDNA. The generated product double-stranded cDNA from each well (containing an index sequence unique to each single-cell reaction) was pooled and subjected to PCR cleanup. After pooling and cleanup, the double-stranded cDNA was split into two reactions to generate separate sequencing libraries for (1) sequence difference analysis and (2) TCR profiling.

[0188] Preparation of libraries for sequence difference analysis Preparation of sequencing libraries for sequence difference analysis was performed by 5'-end capture. Briefly, TnRP1 and TnPR2 transposons and Tn5 transposase were used to tagment the product double-stranded cDNA. Tagmentation resulted in product double-stranded cDNA fragments with intact 5' ends (i.e., 5' ends that retained the index and pre-amplification PCR primer binding site sequences) and tagmented 3' ends that introduced the post-tagmentation amplification primer binding domain. was obtained. The tagmented 5' fragments were amplified and sequencing adapter sequences were introduced using two 5' end capture primers: the first primer ("5' end capture primer 1") hybridizing to the pre-amplification PCR primer binding site sequence, and the second primer ("5' end capture primer 2") hybridizing to the post-tagmentation amplification primer binding domain. The first primer ("5' end capture primer 1") contained P7, i7, Read2, and SMART sequences. The second primer ("5' end capture primer 2") contained P5, i5, and Tn Read1 sequences. After amplification, the library was completed and ready for sequencing.

[0189] Preparation of libraries for TCR profiling Preparation of TCR-specific libraries for TCR profiling analysis was performed in tubes using TCR-specific amplification and addition of sequence adapters. TCR-specific amplification was performed using SMART Primer1 (containing Read2 and SMART sequences) and primers specific for the human TCR alpha / beta chain constant region ("TCRa / b Human Primer1"). In the first round of amplification ("PCR 1"), SMART Primer1 hybridized to the pre-amplification PCR primer binding site sequence and TCRa / b Human Primer1 hybridized to the TCR alpha / beta chain constant region. The second round of amplification ("PCR2") was performed using TCR Primer2 Forward HT Index (hybridized to the Read2 sequence and introduces the P7 and i7 sequences) and TCR a / b Human Primer2 Reverse HT Index (hybridized to the amplified TCR alpha / beta constant region sequence and introduces the Read1, i5 and P5 sequences). After amplification, the library was completed and ready for sequencing.

[0190] Example 2: Single cell T cell receptor profiling Single cells were sorted into lysis buffer in individual wells of a 96-well plate. Reverse transcription reagents were then added to the plate, which contained unique indexed SMART-Seq oligonucleotides per row (A-H, FIG. 7). RT and pre-amplification PCR steps were performed in each well of the plate, and the products were pooled per column, resulting in 12 pools, each containing a mixture of cDNA molecules barcoded according to the individual cell from which they originated. Subsequent library construction steps (as described in more detail below) were performed on each of the 12 pools, reducing the number of libraries that needed to be prepared from 96 to 12. The inclusion of barcodes allows for demultiplexing of the sequencing data in each pool, thereby allowing for resolution of single cells and pairing of sequence information for the TCR-α and TCR-β subunits.

[0191] An overall schematic showing the overall process used in preparing a library for TCR profiling from a single T cell is shown in Figure 6. The process used in preparing the library was similar to the TCR profiling library preparation portion of the process described in Example 1.

[0192] Briefly, single cells were sorted into 96-well plates. Synthesis of first-strand cDNA was primed with a TCR dT primer and performed by MMLV-derived reverse transcriptase (RT). Upon reaching the 5' end of each mRNA molecule, the RT added non-template nucleotides to the first-strand cDNA. SMART-Seq® Indexed Oligonucleotides, which contain row-specific index sequences plus sequences complementary to the non-template nucleotides added by the RT, were hybridized to the first-strand cDNA. In the template switching step, the RT used the SMART-Seq® as a template to introduce additional sequences to the ends of the first-strand cDNA. The remainder of the indexed oligonucleotide was used.

[0193] Each well then underwent a pre-amplification PCR step to generate double-stranded cDNA that served as starting material for PCR amplification and library construction. After a clean-up step, the cDNA from each column of the 96-well plate was pooled into separate tubes (see FIG. 7) and probed with a primer ("SMART TCR-specific sequences were amplified by PCR using a primer ("TCRa / b Human Primer1") complementary to the constant region(s) of TCR-α and / or TCR-β subunits. A round of PCR was then performed to further amplify the variable regions of TCR-α and / or TCR-β subunits and introduce adapter sequences using TCR Primer2 Forward HT Index and TCR a / b Human Primer2 Reverse HT Index. Primers contained adapter and index sequences compatible with the Illumina® sequencing platform (read2+i7+ P7 and read1+i5+P5, respectively). After purification, size selection and quality analysis, the TCR cDNA library was sequenced on the Illumina platform using 300 bp paired-end reads.

[0194] To test workflow performance, TCR-α and TCR-β sequence libraries were generated from either single Jurkat cells or a single cell equivalent of Jurkat RNA (2.5 pg of RNA). Eight cells and eight RNA samples were processed separately in a 96-well plate with different SMART-Seq Indexed Oligos for each type of input. RT and pre-amplification PCR reactions were performed in each well, and cDNA products from the same type of input were pooled together before the next PCR round, as described above. The final libraries were sequenced on a MiSeq using 300 bp paired-end reads. After sequencing, the sequencing data was demultiplexed using barcode sequences derived from the SMART-Seq Indexed Oligos and then analyzed using MiXCR (Bolotin et al., (2015) Nat. Methods 12(5):380-381).

[0195] The percentage of sequencing reads that mapped to the CDR3 region of TCR-α or TCR-β from each cell or RNA sample was determined (Figure 8). For each RNA sample, >96% of the reads mapped to either TCR subunit. In seven of eight cells analyzed, >89% of the reads mapped to either TCR subunit.

[0196] The percentage of sequencing reads from each cell or RNA sample that mapped to the expected Jurkat clonotypes (TRAV8-4, TRAJ3 / TRBV12-3, TRBJ1-2) was also determined (Figure 9). For each RNA sample, the correct Jurkat clonotype was identified in >92% of the reads. Consistent with the read alignment data, the correct Jurkat clonotype was identified in >86% of the reads in 7 of 8 cells analyzed.

[0197] In summary, testing of Jurkat cells in a 96-well format assay approach demonstrated that, on average, 92% of sequencing reads mapped to TCR sequences, while an average of 90% of reads identified the correct Jurkat clonotype.

[0198] Example 3: Single-cell T cell receptor profiling using the ICELL8 system a. Two primer-mediated cDNA synthesis Single cell TCR profiling was also tested in a multi-sample nanodispenser (MSND) configuration. Cells and reagents were dispensed in nanoliter volumes into the wells of an ICELL8® chip using a WaferGen (Fremont, Calif.) ICELL8® MSND system. The workflow is generally outlined in FIG. 10.

[0199] The on-chip reaction process included three dispense steps. In "dispense #1" (single cell solution), MSND was used to dispense T cells with (or without) preprinted barcoded PCR primers onto WaferGen 72x72 chips using a method designed to maximize single cell yield as determined by Poisson statistics. Automated imaging of cells was performed using CellSelect® software (Wafergen, Fremont CA). Wells containing single cells were downselected so that each barcode was used only once. Each barcode (n=1,728) on the chip was present in three copies, resulting in a well-specific address. Cells were lysed on-chip by freeze-thawing and immediately processed.

[0200] In "aliquot #2" (RT mix), first strand cDNA synthesis was primed by a TCR dT primer and performed by MMLV-derived reverse transcriptase (RT) in the presence of cell lysis buffer. Upon reaching the 5' end of each mRNA molecule, the RT added non-template nucleotides to the first strand cDNA. The SMARTSeqv4 Oligonucleotide, containing a sequence complementary to the non-template nucleotide added by the RT, hybridized to the first strand cDNA. In the template switching step, the RT utilized the remainder of the SMART-Seq v4 template switching oligo as a template to introduce additional sequences to the ends of the first strand cDNA.

[0201] Aliquot #3 (preamplification mix) was subjected to 10 cycles of preamplification to introduce the preprinted PCR1-A Primer (in combination with the TCR dT Primer). The chip contents were extracted using a fixture and centrifuged prior to column purification.

[0202] PCR amplification reactions were performed in tubes. The full-length variable region of TCR cDNA was selectively amplified by PCR using an indexed primer (TCR Primer2 Forward HT Index) complementary to the oligonucleotide template sequence and a primer (TCR α / β Human Primer1) complementary to the constant region(s) of TCR-α and / or TCR-β subunits. Subsequent rounds of PCR were performed using TCR Primer2 Forward HT Index and TCR a / b Human Primer2 Reverse HT Index to further amplify the variable regions of TCR-α and / or TCR-β subunits and introduce adapter sequences. Primers contained adapter and index sequences compatible with Illumina sequencing platforms (Read2+i7+ P7 and Read1+i5+P5, respectively). After purification, size selection and quality analysis, the TCR cDNA library was sequenced on the Illumina platform using 300 bp paired-end reads.

[0203] To evaluate the performance of the SMARTer® cDNA Synthesis Kit (Takara Bio USA, Mountain View, CA) in an on-chip workflow, sequencing data generated using the ICELL8® MSND system were analyzed for three independent library preparation runs. The protocol was first performed with Jurkat cells and an RNA control sample, and libraries were generated using a single barcode ((1) and (2)). It was then validated on a preprinted chip containing 1,728 barcodes. The resulting cDNA libraries were sequenced to identify The results were analyzed using MiXCR (Bolotin et al., (2015) Nat. Methods 12(5):380-381).

[0204] In validation experiments using a single barcode, 1,471 Jurkat cells and 6 or 48 PBMC RNA control wells were included in the final library. In both of these experiments, the number of reads that mapped to the CDR3 region of TCR-α or TCR-β was good (~84% in run 1 (1) and ~69% in run 2 (2)), and the majority of these reads were used for clonotype calling. In (1), more than 99% of the reads used for clonotype calling identified the correct Jurkat clonotypes (TRAV8-4, TRAJ3 / TRBV12-3, TRBJ1-2). In (2), where more PBMC RNA control samples were used, this value was ~95% (the remaining ~5% reads identified alternative clonotypes present in the PBMC RNA control).

[0205] We then tested the workflow on an ICELL8® MSND chip with 1,728 preprinted barcodes. In this experiment, 824 Jurkat cells, 10 Jurkat RNA controls, and 10 PBMC controls were included in the final library. In this case, approximately 67% of the reads were mapped to the CDR3 region of TCR-α or TCR-β. More than 99% of the reads used for clonotype calling identified the correct Jurkat clonotype (TRAV8-4, TRAJ3 / TRBV12-3, TRBJ1-2) with good representation of both alpha and beta chains. A summary of this data is shown in the table below.

[0206] [Table 1]

[0207] We further analyzed the alignment of sequencing reads from a single Jurkat cell. We analyzed the sequencing data of 25 randomly selected cells from the cells included in the above experiment and found that the CDR3 regions of TCR-α or TCR-β were not mapped to the TCR-α or TCR-β CDR3 regions. The percentage of sequencing reads that matched the TCR-α or TCR-β sequence was calculated. The results of this analysis are shown in Figure 11, where "R" refers to row position and "C" refers to column position for each cell. In the majority of cells (21 / 25), >60% of the reads mapped to TCR-α or TCR-β sequences. In both cells, the correct Jurkat clonotypes (TRAV8-4, TRAJ3 / TRBV12-3, TRBJ1-2) were identified. Data from control wells yielded a similar range of alignment rates.

[0208] The results above show that single-cell TCR profiling in the MSND format allows the generation of sequencing libraries of approximately 1,000 cells at a time, which can be pooled together and analyzed neatly in a single run (e.g., a single run on a MiSeq®). Using this approach to analyze individual Jurkat cells, we found that in the majority of cells, >60% of the reads mapped to TCR sequences, and approximately 70-80% of these reads were used for clonotype identification.

[0209] b. Single primer-mediated synthesis of cDNA 12A-12D illustrate a variation of the above example in which a single primer is used to generate cDNA using the ICELL8® MSND system, and the cDNA is used according to an embodiment of the invention. As in Example 3a above, cells and reagents are dispensed in nanoliter volumes into the wells of an ICELL8® MSND chip using a WaferGen (Fremont, Calif.) ICELL8® MSND system.

[0210] The on-chip reaction process shown in FIG. 12A includes three dispense steps. In "Dispense #1" (single cell solution), the MSND dispenser dispenses T cells into WaferGen 72×72 SmartChip chips containing preprinted barcoded TSOs using a method designed to maximize single cell yield as determined by Poisson statistics. The barcoded TSOs can contain one or more primer binding sites and a barcode sequence. The barcode sequence can be a well barcode sequence indicating that the TSOs originate from the same well of the 72×72 SmartChip. CellSelect® software performs automated imaging of the cells. Wells containing single cells are downselected so that each barcode is used only once. The cells are lysed on-chip by freeze-thawing and immediately processed.

[0211] In "aliquot #2" (RT-PCR mix), synthesis of first strand cDNA is primed by an oligo-dT primer and carried out by MMLV-derived reverse transcriptase (RT) in the presence of cell lysis buffer. Upon reaching the 5' end of each mRNA molecule, the RT adds non-template nucleotides to the first strand cDNA. A preprinted TSO, containing sequences complementary to the non-template nucleotides added by the RT, was hybridized to the first strand cDNA. In the template switching step, the RT uses the remainder of the TSO as a template to introduce additional sequences to the ends of the first strand cDNA.

[0212] In the illustrated protocol, the terminal sequences of the TSO and CDS are the same to allow synthesis of double-stranded cDNA in a single round of primer amplification. The TSO may contain one or more additional TSO-specific sequences, which can be, for example, primer binding sites, UMIs, cell / sample barcodes, etc. A full-length cDNA library is generated (as shown in the bioanalyzer waveform in Figure 12B) by amplifying the cDNA while still on the chip, using a single primer. The single primer can be a new primer or the TSO and CDS primers used in reverse transcription.

[0213] As shown in Figure 12C, the samples are removed from the chip and pooled in the "off-chip" portion of the protocol. The pooled sample may then be split to generate two samples. One sample may be used to generate an immune cell receptor repertoire sequencing library. The other sample may be used for WTA or other gene-specific amplification.

[0214] As illustrated, samples for generating immune cell receptor repertoire libraries may be amplified using TCR alpha / beta gene-specific primers and TSO end-specific primers. A second nested PCR amplification may also be performed. As shown in the bioanalyzer trace in Figure 12D, amplification can generate libraries containing both TCR alpha and beta receptors.

[0215] In an example implementation of the above protocol, two chips were dispensed with different T cell lines. Chip 1 had 188 TALL-104 cells, two positive control wells (Jurkat RNA-5 pg / well) and two negative control wells. Chip 2 had 94 CCRF-CEM cells, 94 CCRF-CEM cells treated with PMA, two positive control wells (Jurkat RNA-5 pg / well) and two negative control wells.

[0216] Cells were sorted using ICELL8® MSND system and the TCR amplification method was performed as described above. The library was sequenced and analyzed. As shown in FIG. 13, TCRα and TCRβ receptors could be identified in the CCRF and TALL cells used in the experiment. In addition, CCRF cells treated with PMA showed an increase in α and β receptor reads, indicating that the method can detect receptors in activated cells. As shown in FIG. 13, the MiXCR output data was filtered using Excel to determine a higher read threshold than the 1×PBS negative control for both TCRα and TCRβ clonotypes (solid line indicates 80 read counts). Data was analyzed using JMP software and plotted in a box plot, where the lower bar indicates the 25th percentile and the upper bar indicates the 75th percentile. Each point indicates the TCRα and TCRβ clonotypes in the cells. On-chip negative controls were used to set thresholds that allowed for differentiation of samples from NTC or "junk" low-read clonotypes. Data generated from unstimulated CCRF-CEM cells and Jurkat RNA (positive control) and stimulated CCRF-CEM cells and Jurkat RNA are shown. Induction of CCRF-CEM cells with phorbol myristate acetate (PMA) increases expression of the TCRα gene. CCRF-CEM data were generated using cells that were or were not treated with PMA. Treatment of CCRF-CEM cells with PMA improved the call rate by 4-fold (from 14% to 52%) based on data for TCRα and TCRβ clonotypes.

[0217] Figures 14A and 14B show successful whole transcriptome amplification (WTA) of samples split from the initial amplified double stranded cDNA sample generated according to the protocol of Figures 12A-12D. The whole transcriptome amplicons (WTA) generated by splitting the amplification reaction were treated with the NEXTERA™ Nucleic Acid Library Preparation Kit (Illumina) to fragment the amplicons and add adapter sequences. The 5' ends of the amplicons were amplified using primers specific to the TSO specific sequence (e.g., primer binding site) and a primer specific to the Nextera-introduced adapter. The 5' differential expression library was sequenced to determine gene body coverage and major gene components. As shown in Figure 14A and B, this 5'DE library preparation method was able to capture the 5' ends of the amplicons well, as well as identify different cell types.

[0218] Notwithstanding the appended claims, the present disclosure is also defined by the following appended claims. 1. A method for preparing an expression library and an immune cell receptor repertoire library from a ribonucleic acid (RNA) sample, comprising: (a) generating product double-stranded cDNA from an RNA sample using a template-switching reverse transcription reaction; (b) dividing the resulting double-stranded cDNA into a first reaction mixture and a second reaction mixture; (c) end-capturing the product double-stranded cDNA to generate an expression library from the first reaction mixture, and amplifying immune cell receptor-specific cDNA from the product double-stranded cDNA to generate an immune cell receptor repertoire library from the second reaction mixture. A method comprising: 2. The method according to claim 1, wherein end capture and amplification are performed simultaneously. 3. The method according to claim 1, in which end capture and amplification are performed sequentially. 4. The method according to any one of Appendices 1 to 3, wherein the RNA sample is obtained from a cell sample. 5. The method according to any one of appendices 1 to 3, wherein the RNA sample is obtained from a single cell.

[0219] 6. The method of claim 5, wherein the single cell is a T cell. 7. The method of claim 5, wherein the single cell is a B cell. 8. The method of any of claims 5 to 7, further comprising pooling product double-stranded cDNA generated from multiple single-cell RNA samples prior to partitioning. 9. The method of any one of Appendices 5 to 8, wherein a primer or oligonucleotide used in generating the product double-stranded cDNA comprises an indexing sequence that identifies the single cell from which the product double-stranded cDNA was generated. 10. The method of claim 9, wherein the primer or oligonucleotide is a template switching oligonucleotide.

[0220] 11. The method according to any one of appendix 5 to 10, wherein a single cell is obtained from a biological sample. 12. The method of claim 11, further comprising isolating a single cell using a cell sorter. 13. The method according to claim 12, wherein the cell sorter is a flow cytometer. 14. The method of claim 12, wherein the cell sorter is a multi-well based system. 15. The method according to any one of appendices 1 to 14, wherein the expression library is a 5'-end library.

[0221] 16. The method according to any one of appendices 1 to 14, wherein the expression library is a 3'-end library. 17. The method according to any one of appendices 1 to 16, further comprising sequencing the expression library and the immune cell receptor repertoire library. 18. The method of claim 17, further comprising differential expression analysis of a sequenced expression library. 19. The method according to claim 18, wherein the differential expression analysis comprises whole transcriptome analysis (WTA). 20. The method of claim 18 or 19, wherein the differential expression analysis comprises targeted expression analysis.

[0222] 21. The method of claim 20, wherein the target expression analysis includes immune gene expression analysis. 22. Immune cell receptor-specific cDNA contains the 5' end of the immune cell receptor sequence. 22. The method according to any one of Appendices 1 to 21. 23. The method according to any one of appendices 1 to 22, wherein the immune cell receptor-specific cDNA comprises a full-length immune cell receptor sequence. 24. The method of any one of appendices 1 to 23, wherein the amplification step comprises contacting the second reaction mixture with a 5' amplification primer and an immune cell receptor-specific amplification primer. 25. The method of claim 24, wherein the immune cell receptor-specific amplification primers specifically hybridize to the constant region of one or more chains of the immune cell receptor.

[0223] 26. The method of claim 25, wherein the immune cell receptor is a T cell receptor (TCR). 27. The method of claim 25 or 26, wherein the one or more chains is a TCR-α chain, a TCR-β chain or both. 28. The method of claim 25, wherein the immune cell receptor is a B cell receptor (BCR). 29. The method of claim 25 or 28, wherein one or more chains are immunoglobulin chains. 30. The method of any of claims 24 to 29, wherein the immune cell receptor-specific amplification primers comprise one or more sequencing platform adaptor constructs.

[0224] 31. The method of any one of appendices 24 to 30, wherein the 5' amplification primer comprises an indexing sequence. 32. The method of any one of claims 24 to 31, wherein the 5' amplification primer comprises one or more sequencing platform adapter constructs. 33. The method of any of claims 1 to 32, wherein end capture comprises contacting the first reaction mixture with an end amplification primer. 34. The method of claim 33, wherein end capture comprises 5' end capture. 35. The method of claim 34, wherein 5' end capture comprises contacting the first reaction mixture with a 5' end amplification primer.

[0225] 36. The method of claim 35, wherein the 5' terminal amplification primer comprises one or more sequencing platform adapter constructs. 37. The method of claim 33, wherein end capture comprises 3' end capture. 38. The method of claim 37, wherein 3' end capture comprises contacting the first reaction mixture with a 3' end amplification primer. 39. The method of claim 38, wherein the 3' terminal amplification primer comprises one or more sequencing platform adapter constructs. 40. The method of any one of appendices 1 to 39, wherein end capture comprises tagmentation to add a post-tagmentation amplification primer binding domain to the product double-stranded cDNA.

[0226] 41. The method of claim 40, wherein tagmentation comprises amplifying the product double-stranded cDNA using a terminal amplification primer and a post-tagmentation amplification primer that hybridizes to a post-tagmentation amplification primer binding domain. 42. The method of claim 40 or 41, wherein tagmentation further comprises contacting the product double-stranded cDNA with Tn5 transposase. 43. Producing a product double-stranded cDNA comprises: An RNA sample; a first strand complementary deoxyribonucleic acid (cDNA) primer; a template switch oligonucleotide comprising a 3' hybridization domain and a 5' adaptor sequence binding domain; Reverse transcriptase and dNTPs and 43. The method of any one of claims 1 to 42, comprising combining in a reaction mixture. 44. The method of any one of claims 1 to 43, further comprising sequencing genomic DNA (gDNA) of a single cell. 45. The method of claim 44, further comprising isolating gDNA from a single cell prior to sequencing.

[0227] 46. ​​The method of claim 44 or 45, wherein sequencing gDNA of a single cell comprises immune locus-specific sequencing. 47. The method of claim 46, wherein the immune locus-specific sequencing includes T cell receptor (TCR) locus-specific sequencing. 48. The method of claim 46 or 47, wherein the immune locus-specific sequencing comprises B cell receptor (BCR) locus-specific sequencing. 49. A method according to any one of claims 44 to 48, wherein sequencing gDNA from a single cell comprises whole genome sequencing. 50. The method according to any one of appendices 1 to 49, wherein the expression library is a full-length expression library.

[0228] 51. A 5' amplification primer; an immune cell receptor specific amplification primer; Terminal amplification primers and Kit including: 52. The kit of claim 51, further comprising a poly(dT) primer. 53. The kit of claim 51 or 52, wherein the 5' amplification primer comprises an indexing sequence. 54. The kit of any one of appendices 51 to 53, wherein the 5' amplification primer comprises one or more sequencing platform adaptor constructs. 55. A kit according to any one of appendices 51 to 54, wherein the immune cell receptor-specific amplification primer comprises an indexing sequence.

[0229] 56. A kit according to any of appendices 51 to 55, wherein the immune cell receptor-specific amplification primers comprise one or more sequencing platform adaptor constructs. 57. A kit according to any one of appendices 51 to 56, wherein the terminal amplification primer comprises an indexing sequence. 58. A kit according to any one of appendices 51 to 57, wherein the terminal amplification primer comprises one or more sequencing platform adapter constructs. 59. The kit according to any one of appendices 51 to 58, wherein the terminal amplification primer is a 5' terminal amplification primer. 60. A kit according to any one of appendices 51 to 58, wherein the terminal amplification primer is a 3' terminal amplification primer.

[0230] 61. A kit according to any one of appendices 51 to 60, further comprising one or more components for performing a template switching reverse transcription reaction, the component comprising a template switching oligonucleotide comprising a 5' adapter sequence. 62. The 5' amplification primer comprises a 5' adapter sequence or its complement. The kit according to claim 1, 63. The kit of claim 61 or 62, wherein the terminal amplification primer comprises a 5' adapter sequence or its complement. 64. A kit according to any of appendices 51 to 63, comprising a second 5' amplification primer comprising a sequence identical to the 5' portion of the 5' amplification primer. 65. A kit according to any of appendices 51 to 64, comprising a second immune cell receptor-specific amplification primer comprising a sequence identical to a portion of the immune cell receptor-specific amplification primer.

[0231] 66. The kit of claim 65, wherein the portion is a 5' portion of an immune cell receptor-specific amplification primer. 67. The kit of claim 65, wherein the portion is a 3' portion of an immune cell receptor-specific amplification primer. 68. A kit according to any one of appendices 51 to 67, further comprising one or more components for carrying out a tagmentation reaction. 69. One or more components for carrying out a tagmentation reaction include: A transposon nucleic acid comprising a post-tagmentation amplification primer binding domain; a post-tagmentation amplification primer that hybridizes to the post-tagmentation amplification primer binding domain; Transposase or A combination of these 69. The kit of claim 68, comprising: 70. The kit of claim 69, wherein the transposase is Tn5 transposase.

[0232] 71. A method for preparing an immune cell receptor repertoire library from a plurality of single cells, comprising: (a) isolating an RNA sample from each of a plurality of single cells; (b) generating indexed product double-stranded cDNA from the RNA sample using a template-switching reverse transcription reaction; (c) pooling the generated indexed product double-stranded cDNA; (d) amplifying the immune cell receptor-specific cDNA of the indexed product double-stranded cDNA to generate an immune cell receptor repertoire library containing single cell indexed immune cell receptor-specific cDNA; The method includes: 72. The method of claim 71, wherein the single cell is a T cell. 73. The method of claim 71, wherein the single cell is a B cell. 74. The method of any of claims 71 to 73, wherein the primer or oligonucleotide used in generating the indexed product double-stranded cDNA comprises an indexing sequence that identifies a single cell. 75. The method of claim 74, wherein the primer or oligonucleotide is a template switching oligonucleotide.

[0233] 76. The method of claim 74, wherein the primer or oligonucleotide is a 5' amplification primer that contains a sequence identical to the 5' portion of the template switching oligonucleotide used in the template switching reverse transcription reaction. 77. The method of any of claims 71 to 76, wherein the indexed immune cell receptor-specific cDNA comprises the 5' end of the immune cell receptor sequence. 78. The method of any of claims 71 to 77, wherein the indexed immune cell receptor-specific cDNA comprises a full-length immune cell receptor sequence. 79. The method of any of claims 71 to 78, wherein the amplification comprises contacting the pooled indexed product double-stranded cDNA with a 5' amplification primer and an immune cell receptor-specific amplification primer. 80. The method of claim 79, wherein the immune cell receptor-specific amplification primers specifically hybridize to the constant region of one or more chains of the immune cell receptor.

[0234] 81. The method of claim 80, wherein the immune cell receptor is a T cell receptor (TCR). 82. The method of claim 81, wherein the one or more chains are a TCR-α chain, a TCR-β chain, or both. 83. The method of claim 80, wherein the immune cell receptor is a B cell receptor (BCR). 84. The method of claim 83, wherein one or more of the chains is an immunoglobulin chain. 85. The method of any of claims 79 to 84, wherein the immune cell receptor-specific amplification primers comprise one or more sequencing platform adaptor constructs.

[0235] 86. The method of any one of appendices 79 to 85, wherein the 5' amplification primer comprises an indexing sequence. 87. The method of any one of claims 79 to 86, wherein the 5' amplification primer comprises one or more sequencing platform adapter constructs. 88. A method according to any one of claims 79 to 87, in which a single cell is obtained from a biological sample. 89. The method of claim 88, further comprising isolating a single cell using a cell sorter. 90. The method according to claim 89, wherein the cell sorter is a flow cytometer.

[0236] 91. The method of claim 89, wherein the cell sorter is a multi-well based system. 92. The method of any one of claims 71 to 91, further comprising sequencing genomic DNA (gDNA) of a single cell. 93. The method of claim 92, further comprising isolating gDNA from a single cell prior to sequencing. 94. The method of claim 92 or 93, wherein sequencing the gDNA of a single cell comprises immune locus-specific sequencing. 95. The method of claim 94, wherein the immune locus-specific sequencing includes T cell receptor (TCR) locus-specific sequencing.

[0237] 96. The method of claim 94 or 95, wherein the immune locus-specific sequencing comprises B cell receptor (BCR) locus-specific sequencing. 97. A method according to any one of claims 92 to 96, wherein sequencing the gDNA of a single cell includes whole genome sequencing. 98. The method of any one of claims 71 to 97, further comprising sequencing the immune cell receptor repertoire library. a template switching oligonucleotide comprising a 99.5' adaptor sequence; a 5' amplification primer comprising a 5' adapter sequence; Immune cell receptor specific amplification primers and Kit including: 100. The kit of claim 99, wherein the template switching oligonucleotide comprises an indexing sequence.

[0238] 101. The kit of claim 99 or 100, wherein the 5' amplification primer comprises an indexing sequence. 102. The kit of any of appendix 99 to 101, wherein the 5' amplification primer comprises one or more sequencing platform adaptor constructs. 103. The kit of any one of Appendices 99 to 102, wherein the immune cell receptor-specific amplification primer comprises an indexing sequence. 104. A kit according to any of appendices 99 to 103, wherein the immune cell receptor-specific amplification primers comprise one or more sequencing platform adaptor constructs. 105. The kit according to any one of Appendices 99 to 104, comprising a poly(dT) primer.

[0239] 106. A kit according to any one of appendices 99 to 105, further comprising, in addition to the template switching oligonucleotide, one or more components for carrying out a template switching reverse transcription reaction. 107. The kit of any of appendices 99 to 106, comprising a second 5' amplification primer. 108. The kit of claim 107, wherein the second 5' amplification primer comprises one or more sequencing platform adapter constructs. 109. The kit of claim 107 or 108, wherein the second 5' amplification primer comprises a sequence identical to the 5' portion of the 5' amplification primer. 110. The kit of any of appendices 107 to 109, further comprising a third 5' amplification primer.

[0240] 111. The kit of claim 110, wherein the third 5' amplification primer comprises one or more sequencing platform adapter constructs. 112. The kit of claim 110 or 111, wherein the third 5' amplification primer comprises a sequence identical to the 5' portion of the second 5' amplification primer. 113. The kit of claim 107 or 108, wherein the second 5' amplification primer comprises a sequence that hybridizes to a primer binding site present in a 5' non-template sequence bound to the 5' amplification primer. 114. A kit according to any of appendices 99 to 113, comprising a second immune cell receptor-specific amplification primer comprising a sequence identical to a portion of the immune cell receptor-specific amplification primer. 115. The kit according to any one of appendices 99 to 114, wherein the portion is a 5' portion of an immune cell receptor-specific amplification primer.

[0241] 116. The kit according to any one of appendices 99 to 114, wherein the portion is the 3' portion of an immune cell receptor-specific amplification primer.

[0242] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of this invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0243] Thus, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations which, although not explicitly described or shown herein, embody the principles of the present invention and are within its spirit and scope. Moreover, all examples and specific terminology shown herein are intended, in principle, to aid the reader in understanding the principles and concepts of the present invention which the inventors have contributed to the advancement of the art, and are not intended to be limiting to the examples and conditions so specifically shown. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

[0244] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to the filing date of U.S. Provisional Application No. 62 / 459,858, filed February 16, 2017, the disclosure of which is incorporated herein by reference.

Claims

1. a 5' amplification primer; an immune cell receptor specific amplification primer; Terminal amplification primers and Including the kit.

2. The kit of claim 1, further comprising a template switching oligonucleotide comprising a 5' adapter sequence and an indexing sequence that identifies a single cell.

3. further comprising one or more components for performing a tagmentation reaction; The one or more components are A transposon nucleic acid comprising a post-tagmentation amplification primer binding domain; a post-tagmentation amplification primer that hybridizes to the post-tagmentation amplification primer binding domain; Transposase, or A combination of these The kit of claim 1 , comprising:

4. a template switching oligonucleotide comprising a 5′ adaptor sequence and a single cell identifying indexing sequence; a 5' amplification primer comprising the 5' adapter sequence; Immune cell receptor specific amplification primers and Including the kit.

5. The kit of claim 1 or 4, wherein the 5' amplification primer and the terminal amplification primer each comprise one or more sequencing platform adaptor constructs.

6. The kit of claim 1 or 4, wherein the immune cell receptor is a T cell receptor (TCR) or a B cell receptor (BCR).

7. The kit of claim 1 or 4, further comprising a poly(dT) primer.

8. The kit of claim 1 or 4, further comprising at least one polymerase.

9. The kit of claim 8 , wherein the at least one polymerase is a reverse transcriptase.

10. The kit of claim 1 or 4, further comprising dNTPs.

11. 1. A method for preparing an expression library and an immune cell receptor repertoire library from a ribonucleic acid (RNA) sample obtained from a single cell, comprising: (a) generating product double-stranded cDNA from an RNA sample obtained from a single cell using a template switching reverse transcription reaction; (b) partitioning the generated product double-stranded cDNA into a first reaction mixture and a second reaction mixture; (c) end-capturing the product double-stranded cDNA to generate an expression library from the first reaction mixture, and amplifying immune cell-specific cDNA from the product double-stranded cDNA to generate an immune cell receptor repertoire library from the second reaction mixture. A method comprising:

12. 12. The method of claim 11, wherein a primer or oligonucleotide used in generating the product double-stranded cDNA comprises an indexing sequence that identifies the single cell from which the product double-stranded cDNA was generated.

13. 13. The method of claim 12, further comprising pooling the product double-stranded cDNA generated from the multiple single-cell RNA samples prior to partitioning in (b).

14. 1. A method for preparing an immune cell receptor repertoire library from a plurality of single cells, comprising: (a) isolating an RNA sample from each of the plurality of single cells; (b) generating indexed product double-stranded cDNA from said RNA sample using a template-switching reverse transcription reaction; (c) pooling the generated indexed product double-stranded cDNA; (d) amplifying immune cell-specific cDNAs from the indexed product double-stranded cDNA to generate an immune cell receptor repertoire library comprising single cell-indexed immune cell-specific cDNAs; A method comprising:

15. A method of using the kit of claim 1, comprising: (a) amplifying nucleic acid in a sample; (b) partitioning the amplified nucleic acid into a first reaction mixture and a second reaction mixture; (c) contacting the end amplification primers with the nucleic acids of the first reaction mixture to generate an end-trapped expression library; (d) contacting the nucleic acid of the second reaction mixture with the 5' amplification primer and an immune cell receptor-specific amplification primer to generate an immune cell receptor repertoire library; A method comprising:

16. 16. The method of claim 15, wherein the nucleic acid in the sample is obtained from a single cell.

17. A method for using the kit according to claim 4, comprising: (a) performing a template switching reverse transcription reaction using the template switching oligonucleotide and an RNA sample obtained from a single cell to generate an indexed product double-stranded cDNA; (b) pooling the generated indexed product double-stranded cDNA; (c) contacting the pooled indexed product double-stranded cDNA with said 5' amplification primer and an immune cell receptor-specific amplification primer to generate an immune cell receptor repertoire library comprising single cell indexed immune cell-specific cDNA; A method comprising:

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