Compositions and methods for library preparation

Abstract

Library preparation methods for use in high-throughput methods for screening large populations of variant proteins, for instance antibodies, are provided. Approaches include addition of a template switch oligo (TSO) primers to amplicons and nested reverse primers. Such approaches may be suitable for library preparation from RNA samples from organisms having a high numbers of V-genes and J-genes, especially for single-cell enrichment.

Description

OMAB2.012WO PATENTCOMPOSITIONS AND METHODS FOR LIBRARY PREPARATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No.63 / 733,936, filed December 13, 2024, the content of which is incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled OMAB2.012WO.xml, created November 17, 2025, w’hich is approximately 28,565 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUNDField

[0003] The present disclosure generally relates to compositions, kits, methods and systems for nucleic acid sequencing applications.Description of the Related Art

[0004] High-throughput antibody repertoire sequencing (Ig-Seq) in antibody discovery reveals clonal expansion and identifies sequence motifs. Some Ig-Seq heavy chain (IgH) library preparation conventionally requires a large panel of gene-specific primers to cover the full germline sequence space. Due to factors such as primer-primer interactions, mispriming, and differences in annealing temperatures, these primer cocktails introduce systematic biases which may substantially misrepresent the antibody repertoire. Additionally, errors introduced via multiplex polymerase chain reaction (mPCR) library preparation and sequencing result in overestimation of antibody diversity. There are continuing needs to develop improved methods to generate comprehensive antibody repertoires with reliable identification of rare clonotypes.SUMMARY

[0005] In an aspect, the present disclosure provides for a method of preparing a nucleic acid for antibody sequencing, the method including:(a) preamplifying an antibody cDNA sequence to generate a preamplified antibody cDNA sequence by introducing a first forward primer and a first reverse primer to the antibody cDNA sequence, the first forward primer including a template switch oligo (TSO) sequence;(b) amplifying the preamplified antibody cDNA sequence of (a) to generate a first amplicon, the amplifying of (b) including introducing a second forward primer and a second reverse primer, the second forward primer including a TSO sequence and a first priming sequence, the second reverse primer including a second priming sequence; and(c) amplifying the first amplicon of (b), the amplifying of (c) to generate a second amplicon including introducing a third forward primer and a third reverse primer, the third forward primer including a first portion corresponding to a length of the first priming sequence and a second portion including a first clustering sequence, the third reverse primer including at least a first portion corresponding to the first priming sequence and a second clustering sequence.

[0006] In some examples, (a) includes using a plurality of first reverse primers each having different sequences, and the first forward primer includes only SEQ. ID. No. 1.

[0007] In some examples, the first reverse primer binds to the constant region of the antibody cDNA sequence at a first position, the second reverse primer binds to a constant region of the amplified antibody cDNA sequence at a second position, and wherein a 5’ end of the first position is separated from a 5’ end of the second position by a range of from about 49 to about 351 base pairs. In some examples, the 5’ end of the first position is separated from the 5’ end of the second position by about 49, 70, 75, 99, 111, 153, 154, 155, 204, or 351 base pairs.

[0008] In some examples, the method includes reverse transcribing sample RNA using a reverse transcriptase to generate the antibody cDNA sequence, wherein the reverse transcribing adds the TSO sequence to the antibody cDNA sequence. In further examples, thereverse transcribing also adds a unique molecular identifier (UMI) sequence to the antibody cDNA sequence. In some examples, the sample RNA is a rodent RNA sample, a human RNA sample, or a chicken RNA sample. In some examples, the sample RNA is a mouse RNA sample, a rat RNA sample, or a rabbit RNA sample.

[0009] In some examples, the preamplifying of (a) selects for an antibody cDNA oligonucleotide having a sequence corresponding to the TSO forward primer and the first reverse primer. In some examples, the antibody cDNA sequence includes an IgG sequence or an IgM sequence.

[0010] In some examples, one of the first clustering sequence or the second clustering sequence includes a P7 sequence, and wherein the other of the first clustering sequence or the second clustering sequence includes a P5 sequence. In some further examples, the first clustering sequence includes a P7 sequence and wherein the second clustering sequence includes a P5 sequence. In some examples, one of the third forward primer and the third reverse primer includes an indexing sequence. In some examples, the antibody cDNA sequence includes a V(D)J sequence.

[0011] In some examples, the first priming sequence and the second priming sequence are next generation (NGS) priming sequences. In some examples, the first priming sequence and the second priming sequence are sequencing-by-synthesis (SBS) priming sequences. In some examples, one of the first priming sequence or the second priming sequence includes a Read 1 sequence, and wherein the other of the first priming sequence or the second priming sequence includes a Read 2 sequence.

[0012] In some examples, the first SBS priming sequence includes a Read 2 sequence, and wherein the second SBS priming sequence includes a Read 1 sequence.

[0013] In some examples, the antibody cDNA sequence of (a) includes a constant region, the first reverse primer and the second reverse primer including sequences corresponding to portions of the constant region.

[0014] In some examples, the first reverse primer includes one of SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 18, SEQ. ID. No. 21, or SEQ. ID. No. 23. In some examples, the second reverse primer includes one of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ID. No. 17, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No. 22, or SEQ. ID. No. 24. Insome examples, the first reverse primer includes one of SEQ. ID. No. 5 or SEQ. ID. No. 6, and the second reverse primer includes SEQ. ID. No. 10. In some examples, the first reverse primer includes SEQ. ID. No. 7 and the second reverse primer includes SEQ. ID. No. 11. In some examples, the first reverse primer includes SEQ. ID. No. 18 and the second reverse primer includes SEQ. ID. No. 19 or SEQ. ID. No. 20. In some examples, the first reverse primer includes SEQ. ID. No. 21 and the second reverse primer includes SEQ. ID. No. 22. In some examples, the first reverse primer includes SEQ. ID. No. 23 and the second reverse primer includes SEQ. ID. No. 24.

[0015] In some examples, the method includes obtaining an RNA sample from a single cell. In some examples, the method includes obtaining an RNA sample from less than 1000 cells. In some examples, the method includes obtaining an RNA sample from a bulk cell sample.

[0016] In another aspect, the present disclosure provides for a method of determining an antibody sequence, the method including:completing a library preparation method in accordance with the present disclosure to produce a sequencing product; andsequencing the sequencing product.

[0017] In some examples, the step of sequencing includes conducting next generation sequencing (NGS). In some examples, the step of sequencing includes conducting sequencing by synthesis (SBS).

[0018] In another aspect, the present disclosure provides for an amplicon prepared by a library preparation method in accordance with the present disclosure.

[0019] In another aspect, the present disclosure provides for a kit for preparing a nucleic acid for antibody sequencing, the kit including:a first forward primer, the first forward primer including a template switch oligo (TSO) sequence;a first reverse primer;a second forward primer including a TSO sequence and a first priming sequence;a second reverse primer including a second priming sequence;a third forward primer including a first portion corresponding to a length of the first priming sequence and a second portion including a first clustering sequence; and a third reverse primer including at least a first portion corresponding to the first priming sequence and a second clustering sequence.

[0020] In some examples, the kit includes a plurality of first reverse primers having different sequences, and wherein the first forward primer includes only the TSO sequence. In some examples, the first priming sequence and the second priming sequence are next generation (NGS) priming sequences. In some examples, the first priming sequence and the second priming sequence are sequencing-by-synthesis (SBS) priming sequences.

[0021] In some examples, one of the first priming sequence or the second priming sequence includes a Read 1 sequence, and the other of the first priming sequence or the second priming sequence includes a Read 2 sequence. In some examples, the first SBS priming sequence includes a Read 2 sequence, and the second SBS priming sequence includes a Read 1 sequence. In some examples, one of the third forward primer and the third reverse primer includes an indexing sequence.

[0022] In some examples, one of the first clustering sequence or the second clustering sequence includes a P7 sequence, and the other of the first clustering sequence or the second clustering sequence includes a P5 sequence. In some examples, the first clustering sequence includes a P7 sequence and the second clustering sequence includes a P5 sequence. In some examples, the first reverse primer and the second reverse primer include sequences corresponding to portions of a constant region of a target cDNA oligonucleotide.

[0023] In some examples, the first reverse primer includes one of SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 18, SEQ. ID. No. 21, or SEQ. ID. No. 23. In some examples, the second reverse primer includes one of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ID. No. 17, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No. 22, or SEQ. ID. No. 24. In some examples, the first reverse primer includes one of SEQ. ID. No. 5 or SEQ. ID. No. 6, and the second reverse primer includes SEQ. ID. No. 10. In some examples, the first reverse primer includes SEQ. ID. No. 7 and the second reverse primer includes SEQ. ID. No. 11. In some examples, the first reverse primer includes SEQ. ID. No. 18 and the second reverse primer includes SEQ. ID. No. 19 or SEQ. ID. No. 20. In some examples, the first reverse primerincludes SEQ. ID. No. 21 and the second reverse primer includes SEQ. ID. No. 22. In some examples, the first reverse primer includes SEQ. ID. No. 23 and the second reverse primer includes SEQ. ID. No. 24.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a flow chart illustrating a method in accordance with the present disclosure.

[0025] FIG. 2 illustrates amplicons at each step of a first example approach,

[0026] FIG, 3 illustrates amplicons at each step of a second example approach.

[0027] FIG. 4A is a Western blot image of representative Rabbit VH amplicons.

[0028] FIG, 4B is a Western blot image of representative Rabbit VK amplicons.DETAILED DESCRIPTION

[0029] Sequencing human antibodies from transgenic organisms poses several challenges. For example, certain transgenic organisms, particularly rats or mice, have many V- genes and J-genes that can be involved in the V(D)J recombination that occurs in T and B cell maturation. Certain previous approaches have used large primer pools to target all 44 V-genes and 9 J- genes of rats or mice.

[0030] However, certain sequences in traditional amplicon sequencing-by- synthesis (SBS) or next generation sequencing (NGS) library preparation methods with such gene-specific forward and reverse primers can experience amplification bias. See T. A. Khan, et al., Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting, Sci. Adv. 2, e1501371 (2016) (incorporated herein in its entirety). For example, factors such as primer-primer interactions, mispriming, and differences in annealing temperatures, the primer cocktails used for NGS library preparation may introduce systematic biases which may substantially misrepresent the antibody repertoire. Similarly, errors introduced via multiplex polymerase chain reaction (mPCR) library preparation and sequencing result in overestimation of antibody diversity.

[0031] For at least these reasons, certain sequences may be over-represented by SBS sequencing platforms. Additionally, these biases can make rare antibody sequences more difficult to find, especially in situations where the sequences are being enriched from singlecells. When sequences are enriched from single cells, low and / or minimal amounts of RNA may be available per cell.

[0032] The present disclosure provides for approaches of library preparation which incorporate template switch oligo (TSO) primers and multiple rounds of amplification to create semi-nested primers within the final library preparation product. Such approaches can help to avoid amplification biases and reduce the size of the primer pool needed. In some embodiments, the present disclosure provides for methods and kits using a unique molecular identifier (UMI) and universal 5’ adapter, which requires only a single forward primer in lieu of a primer cocktail as described with respect to standard approaches. Further, the addition of a UMI to the DNA sequences during library preparation can advantageously allow for UMI correction analysis. UMI correction analysis can help enable reliable identification of rare clonotypes by minimizing the effect of PCR amplification and sequencing errors in post processing. Approaches using UMI may also help to streamline library preparation by eliminating the intensive optimization and laborious assembly required for standard mPCR approachesMethods of Preparing Sequencing Library

[0033] FIG. 1 is a flow chart illustrating an example method 100 for preparing a sequencing library. The method 100 can be used to prepare nucleic acids for purposes of antibody sequencing.

[0034] At step 102, reverse transcription is optionally conducted. Reverse transcription can incorporate a template switch oligo (TSO) sequence into the resulting cDNA. Reverse transcription can be conducted using a reverse transcriptase to generate an antibody cDNA sequence. The reverse transcribing adds the TSO sequence to the antibody cDNA sequence. Optionally, the reverse transcribing can also add a unique molecular identifier (UMI) sequence to the antibody cDNA sequence. In some examples, the sample RNA is a rodent RNA sample, a human RNA sample, or a chicken RNA sample. In some examples, the sample RNA is a mouse RNA sample, a rat RNA sample, or a rabbit RNA sample.

[0035] At step 104, an antibody cDNA sequence is preamplified. Step 104 may be referred to as PCR0 herein. Step 104 can include introducing a first forward primer and a first reverse primer to the antibody cDNA sequence. The first forward primer can include a TSOsequence. Step 104 can therefore select for products of step 102 that have incorporated TSO sequences. In some examples, step 104 can include using a plurality of first reverse primers each having different sequences. In some examples each first forward primer includes only SEQ. ID. No. 1. In some examples, the first reverse primer includes one of SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 15, SEQ. ID. No. 16, or SEQ. ID. No. 18.

[0036] At step 106, a first amplification is conducted. Step 106 may be referred to as PCR1 herein. The first amplification can incorporate sequencing handles. In some examples, the preamplified antibody cDNA sequence of step 104 can be amplified by introducing a second forward primer and a second reverse primer, the second forward primer including a TSO sequence and a first priming sequence, the second reverse primer including a second priming sequence. In some examples, the second reverse primer includes one of SEQ. ID. No.10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ID. No. 17, SEQ. ID. No. 19, or SEQ. ID. No.20.

[0037] At step 108, a second amplification is conducted. Step 108 may be referred to as PCR2 herein. The second amplification can incorporate first and second clustering sequences. The amplified antibody cDNA sequence of step 106 can be amplified by introducing a third forward primer and a third reverse primer. The third forward primer can include a first portion corresponding to a length of the first priming sequence and a second portion including a first clustering sequence. The third reverse primer includes at least a first portion corresponding to the first priming sequence and a second clustering sequence. The product of step 108 can be used for sequencing. In some examples, one of the first clustering sequence or the second clustering sequence includes a P7 sequence, and the other of the first clustering sequence or the second clustering sequence includes a P5 sequence. In some examples, the first clustering sequence includes a P7 sequence and the second clustering sequence includes a P5 sequence. In some examples, one of the third forward primer and the third reverse primer includes an indexing sequence. In some examples, the first priming sequence and the second priming sequence are next generation (NGS) priming sequences. In some further examples, the first priming sequence and the second priming sequence are sequencing-by-synthesis (SBS) priming sequences. In yet further examples, one of the first priming sequence or the second priming sequence includes a Read 1 sequence, and the otherof the first priming sequence or the second priming sequence includes a Read 2 sequence. In yet further examples, the first SBS priming sequence includes a Read 2 sequence, and the second SBS priming sequence can include a Read 1 sequence.

[0038] In some examples, the antibody cDNA sequence includes a constant region. The first reverse primer and the second reverse primer include sequences corresponding to portions of the constant region. The first reverse primer and the second reverse primer may be capable of binding to the constant region at different positions. The size of amplicons created at each step of method 100 may be a consideration. Certain amplicon sizes may result in greater or lesser amplification. When preparing a library from a limited amount of RNA, it may be desirable to choose first and second reverse primers that create amplicons of a particular size. Amplicon size may be related to the position on the constant region of the antibody cDNA to which the first and second reverse primers bind. Additionally, a semi-nested PCR approach where the reverse primers of PCRO and PCR1 target different portions of the constant region can advantageously provide increased target specificity.

[0039] In some examples, the first reverse primer can be capable of binding to the constant region of the antibody cDNA sequence at a first position. The second reverse primer can be capable of binding to a constant region of the amplified antibody cDNA sequence at a second position. In some embodiments, the second position is closer to the VDJ region than the first position such that the first reverse primer binds at an outer position and the second reverse primer binds at an inner position on the constant region. In some examples, the 5’ end of the first position may be separated from a 5’ end of the second position by 30, 40, 50, 60, 70, 80, 90, 100, 110, 120130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 base pairs, or within a range defined by any two of the previous values, though in some instances other values can be suitably implemented. In some examples, the 5’ end of the first position may be separated from a 5’ end of the second position by a range of 49 to 351 base pairs, though in other values outside this range may be suitably implemented. In some examples, the 5’ end of the first position is separated from the 5’ end of the second position by 49, 70, 75, 99, 111, 153, 154, 155, 204, or 351 base pairs. For example, the following combination of PCRO and PCR1 reverse primers may result in the base pairseparation as indicated below, where the distance between primer pairs is defined by the number of base pairs between the 5’ prime ends of each primer.Species PCR0 primer PCR1 primer Distance between primer pairs (bp) Rat IgG SEQ. ID. No. 5 SEQ. ID. No. 10 351(RatIGHG CH2 rev outer) (RatIGHG rev inner Rl)Rat IgM: SEQ. ID. No. 7 SEQ. ID. No. 11 49(RatIGHM rev outer) (RatIGHM rev inner Rl)Human SEQ. ID. No. 12 SEQ. ID. No. 14 155 VK (hVK_rev_outer1) (hVK_rev_inner_Read1)SEQ. ID. No. 13 SEQ. ID. No. 14 75 (hVK rev outer2) (hVK rev inner Readl)Human SEQ. ID. No. 15 SEQ. ID. No. 17 204 VL (hVL_rev_outer1) (hVL_rev_inner_Read1)SEQ. ID. No. 16 SEQ. ID. No. 17 153 (hVL rev outer2) (hVL rev inner Readl)Mouse SEQ. ID. No. 18 SEQ. ID. No. 19 99 IgG (msIgG_CH1_outer_1) (msIgG_CH1_inner1-1)SEQ. ID. No. 18 SEQ. ID. No. 20 111 (msIgG_CH1_outer_1) (msIgG_CH1_inner1-2)Rabbit SEQ. ID. No. 21 SEQ. ID. No. 22 154 VH (RabbitVH-rev-outer) (RabbitVH rev inner)Rabbit SEQ. ID. No. 23 SEQ. ID. No. 24 70VK (RabbitVK-rev- outer) (RabbitVK rev inner)

[0040] In some examples, the antibody cDNA sequence comprises an IgG sequence or an IgM sequence. In some examples, the antibody cDNA sequence comprising a V(D)J sequence.

[0041] Methods in accordance with the present disclosure may include obtaining an RNA sample from a single cell. In other examples, the method may include obtaining an RNA sample from less than 1, 5, 10, 50, 100, 250, 500, 750, or 1000 cell(s). In some examples, the method may include obtaining an RNA sample from a bulk cell sample. In such examples, the prepared library can include oligonucleotides corresponding to a plurality of the sample cells.

[0042] In other aspects, the present disclosure provides for a method of determining an antibody sequence. The method can include completing the method 100 discussed herein to produce a sequencing product and sequencing the sequencing product. Suitable sequencingmethods may include next generation (NGS) sequencing and / or sequencing by synthesis (SBS).

[0043] Microcapillary arrays have recently been employed in approaches for high- throughput analysis, screening, and protein engineering with large numbers of biological samples, for example in an approach that has been termed “microcapillary single-cell analysis and laser extraction” or “pSCALE”. See Chen et al. (2016) Nature Chem. Biol. 12:76-81; DOI: 10.1038 / NCHEMBIO.1978; see also US Pub. Nos. 2022 / 0162594, 2022 / 0373440, 2020 / 0080075, 2018 / 0188276 (each reference incorporated herein in its entirety). This approach relies on the spatial segregation of single cells within a microcapillary array, and thus enables repeated imaging, cell growth, and protein expression of the separate samples within each microcapillary of the microcapillary array. Accordingly, the technique enables massively parallel, quantitative biochemical and biophysical measurements on millions or multi-millions of samples within a microcapillary array, for example, in the analysis of millions or multimillions of variant proteins (e.g,, variant antibodies) expressed from yeast, bacteria, rodent, human, chicken, or other suitable cells distributed throughout the array. Advantageously, the approach allows for the simultaneous time-resolved kinetic analysis of the multiplexed samples, as well as the sorting of those cells based on targeted phenotypic features. After such cell sorting, library preparation methods of the present disclosure can be used to prepare samples for sequencing.Kit for Preparing DNA Library

[0044] In other aspects, the present disclosure provides for kits to carry out library preparation as discussed with respect to FIG. 1. A kit for preparing a nucleic acid for antibody sequencing can include a first forward primer, the first forward primer including a template switch oligo (TSO) sequence; a first reverse primer; a second forward primer including a TSO sequence and a first priming sequence; a second reverse primer including a second priming sequence; a third forward primer including a first portion corresponding to a length of the first priming sequence and a second portion including a first clustering sequence; and a third reverse primer including at least a first portion corresponding to the first priming sequence and a second clustering sequence.

[0045] In some examples, the kit can include a plurality of first reverse primers having different sequences. The first forward primer may include only the TSO sequence. The first priming sequence and the second priming sequence can be next generation (NGS) priming sequences. In some examples, the first priming sequence and the second priming sequence can sequencing-by-synthesis (SBS) priming sequences. For instance, one of the first priming sequence or the second priming sequence includes a Read 1 sequence, and the other of the first priming sequence or the second priming sequence includes a Read 2 sequence. The first SBS priming sequence can include a Read 2 sequence, and the second SBS priming sequence can include a Read 1 sequence,

[0046] One of the third forward primer and the third reverse primer can include an indexing sequence. Clustering can allow oligonucleotides of a prepared library to bind to a flow cell surface for sequencing. One of the first clustering sequence or the second clustering sequence can include a P7 sequence. The other of the first clustering sequence or the second clustering sequence includes a P5 sequence. The first clustering sequence can include a P7 sequence and the second clustering sequence can include a P5 sequence.

[0047] The first reverse primer and the second reverse primer include sequences corresponding to portions of a constant region of a target cDNA oligonucleotide. The first reverse primer can include one of SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 18, SEQ. ID. No.21, or SEQ. ID. No. 23. The second reverse primer can include one of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ID. No. 17, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No. 22, or SEQ. ID. No. 24. The first reverse primer can include one of SEQ. ID. No. 5 or SEQ. ID. No. 6, and the second reverse primer includes SEQ. ID. No. 10. The first reverse primer can include SEQ. ID. No. 7 and the second reverse primer includes SEQ. ID. No. 11. The first reverse primer includes SEQ. ID. No. 18 and the second reverse primer includes SEQ. ID. No. 19 or SEQ. ID. No. 20. The first reverse primer includes SEQ. ID. No. 21 and the second reverse primer includes SEQ. ID. No. 22. The first reverse primer includes SEQ. ID. No. 23 and the second reverse primer includes SEQ. ID. No. 24.Definitions

[0048] The term “antibody” is used in the broadest sense and includes, for example, an intact immunoglobulin or an antigen binding portion. Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. The term “antibody” can include tetrameric antibodies of two heavy chains and two light chains, as well as antigen binding fragments such as Fv, Fab and scFvs. In some cases, the present disclosure provides for bispecific antibodies that include at least one antigen binding domain as outlined herein.

[0049] Variant proteins and / or variant polypeptides to be analyzed after library preparation in accordance with the present disclosure can include but are not limited to secreted proteins. In some embodiments, the secreted proteins are from a recombinant protein and / or polypeptide library. In some embodiments, the secreted proteins are from a recombinant protein and / or polypeptide library. In some embodiments, the secreted proteins are from a recombinant protein and / or polypeptide library from a mammalian cell line. In some embodiments, the recombinant protein and / or polypeptide library includes full length mammalian antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length mammalian antibodies, including IgGl, IgG2, and IgG4 antibodies and variants thereof. In some embodiments, the recombinant protein and / or polypeptide library includes full length human antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length human antibodies, including IgGl, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length mouse antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length mouse antibodies, including IgGl, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length rat antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length rat antibodies, including IgGl, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes antibody fragments (Fab). In some embodiments, the recombinant protein and / or polypeptide library includes single chain variable fragments (scFv). In some embodiments, the recombinant protein and / or polypeptide library includes natural protein ligands. In some embodiments, the recombinant protein and / or polypeptide library includes natural protein ligands to a defined target proteinand / or polypeptide. In some embodiments, the recombinant protein and / or polypeptide library includes target antibodies and / or fragments thereof. In some embodiments, the recombinant protein and / or polypeptide library’ includes target antibody heavy' chains and / or fragments thereof, such as variable heavy chains. In some embodiments, the recombinant protein and / or polypeptide library includes target antibody light chains and / or fragments thereof, such as variable light chains. In some embodiments, the system of the present invention allows for accurate pairing of VH / VL (variable heavy chains and variable light chains).

[0050] The population of variant proteins is typically generated using a genetic library in a biological expression system, for example in an in vitro (i.e,, cell-free) expression system or in an in vivo or cellular expression system. Exemplary cellular expression systems include, for example, animal systems (e.g., mammalian systems), fungal systems (e.g,, yeast systems), bacterial systems, insect systems, or plant systems. In specific embodiments, the expression system is a mammalian system or a yeast system. In specific embodiments, the expression system is an avian system (for example, a chicken system). The expression system, whether cellular or cell-free, typically includes a library of genetic material encoding the population of variant proteins. Cellular expression systems may offer an advantage that cells with a desirable phenotype, for example cells that express a particular variant protein of interest, such as a variant protein capable of associating with an immobilized target molecule with high affinity, can be grown and multiplied, thus facilitating and simplifying the identification and characterization of the proteins of interest expressed by the cells. In some embodiments, the biological expression system includes a mammalian cell line. In some embodiments, the mammalian cell line is selected from the group consisting of CHO-K1, CHO-S, HEK293T, and / or any derivatives of these cell types. In some embodiments, the mammalian cell line is CHO-K1. In some embodiments, the mammalian cell line is CHO-S. In some embodiments, the mammalian cell line is HEK293T. In some embodiments, the mammalian cell line is selected from the group consisting of human, mouse, and / or rat hybridoma cell lines. In some embodiments, the mammalian cell line is a human hybridoma cell line. In some embodiments, the mammalian cell line is a mouse hybridoma cell line. In some embodiments, the mammalian cell line is a rat hybridoma cell line.

[0051] Genetic libraries encoding large populations of variant proteins are often utilized in systems relying on the process of directed evolution to identify proteins withadvantageous properties, such as high-affinity binding to target molecules, stability, high expression, or particular spectroscopic, e.g., fluorescence, or enzymatic activities. Often the libraries include genetic fusions with sequences from the host expression system, for example fragments of proteins directing subcellular localization, where the expressed population of variant fusion proteins are directed by the targeting fragment to a particular location of the cell or virus particle for purposes of activity screening of the variant protein population. Large numbers of variant proteins (e.g., 106variants, 108variants, 1010variants, 1012variants, or even more variants) can be generated using routine bioengineering techniques, as is well known in the art. Such libraries can include any of the variant proteins described herein, including antibodies, antibody fragments, single chain variable fragments, or natural protein ligands. In some embodiments, the system of the present invention allows for accurate pairing of VH / VL (variable heavy chains and variable light chains).

[0052] Accordingly, in some embodiments, the variant proteins to be sequenced using the present methods are soluble proteins, for example soluble proteins that are secreted by a cellular expression system. Exemplary soluble variant proteins include antibodies and antibody fragments, alternative protein scaffolds, such as disulfide-bonded peptide scaffolds, extracellular domains of cell -surface receptor proteins, receptor ligands, such as, for example, G-protein coupled receptor ligands, other peptide hormones, lectins, and the like. Advantageously, when using a microcapillary array, the variant proteins screened for binding activity do not need to be covalently attached to the cell or virus that expresses them in order to be identified following a screening assay, since a variant protein with a desired binding activity and the cell that expressed it remain co-localized within the same microcapillary of a microcapillary array throughout a screening assay. Isolation of the contents of the desired microcapillary, followed by library preparation in accordance with the present disclosure and / or propagation of the cell or virus clone responsible for expression of the desired variant protein, thereby enables the identification and characterization of that protein. Unlike screening assays where a variant protein of interest is displayed by fusion of the protein to a molecule on the surface of a cell or virus particle, the variant proteins identified in the screening methods need not be altered in any way following their identification. The observed activities of the variant proteins in the screens are thus more likely to represent the actual activities of those proteins m their subsequent applications.

[0053] In other embodiments, however, it may be desirable for the variant proteins to be membrane-associated proteins, for example proteins remaining associated with the surface of a cell or a viral particle in an expression system. Screening of cell-associated variant proteins may be desirable where the variant protein and its target molecule mediate interactions between two cells within a biological tissue. The ability to screen against cell-associated variant proteins may also be desirable in screening for interactions with traditionally “non¬ druggable” protein targets, such as, for example, G-protein coupled receptors or ion channels.

[0054] In addition to a variant protein, each microcapillary in screening methods using microcapillary arrays also includes an immobilized target molecule. The immobilized target molecule serves as the potential binding partner for the variant protein of the screening assay. Unlike the population of variant proteins, where each microcapillary ideally contains a variant protein of slightly different sequence, the immobilized target molecules ideally have the same molecular structure in each microcapillary of the array. In some embodiments, there is no binding or other interaction between the variant protein and another agent or molecule (e.g., the target molecule) prior to the addition of the variant protein to the microcapillary. In some embodiments, the interaction between the variant protein and the target molecule occurs within the microcapillary and / or microcavity.EXAMPLESConstruction of NGS Library

[0055] FIGs, 2 and 3 show working examples of methods for constructing NGS libraries, for example for use on SBS platforms. Such examples are illustrative and are not intended to limit the scope of the present disclosure in any way.Example 1 — Construction of NGS Library from Unsorted Splenocytes

[0056] FIG, 2 shows an example process for constructing an NGS library from unsorted cells, for example splenocytes or lymphocytes. In this example, the library was created using unsorted rat cells. It is to be understood that unsorted cells of other species can be suitable sources of RN samples for NGS library preparation in accordance with the presentdisclosure. For example, the cells can be, among other possible ceil types, rodent cells, for instance mouse or rat cells, or human cells.

[0057] Table 1 below lists example primers used at each step of the method for different types of rat antibody isotypes.Table 1.Reartion Antibody Forward primer Reverse primerisotypeRT Any SEQ. ID No. 1 (dr TSO) SEQ, ID No. 3 (Anchored oligo(dT)30)PCR0 Rat IgG SEQ. ID. No. 4 (TSO F) SEQ. ID. No. 5(RatIGHG CH2 rev outer)Rat IgG SEQ. ID. No. 4 (TSO_F) SEQ. ID No. 6(RatIGHG CH 1 _rev_outer)Rat IgM SEQ. ID. No. 4 (TSO F) SEQ. ID. No. 7(RatIGHM rev outer)PCR1 Rat IgG SEQ. ID. No, 8 (R2- SEQ, ID. No. 10TSO H (RatIGHG_rev_inner_R1)Rat IgM SEQ. ID. No. 8 (R2- SEQ. ID. No. 11TSO F) (RatIGHM rev inner Rl )PCR2 Any index fwd index rev 00#

[0058] With reference to FIG, 2, the step of reverse transcription (RT) is performed on bulk-extracted RNA. Reverse transcriptase synthesizes of cDNA encoding antibody sequences from RNA samples while incorporating the template switching oligo (TSO), RNA sample sequences may include an untranslated region (UTR) sequence, a V(D)J sequence, and a constant sequence. Working dilutions were made for lysis and reverse transcription reactions. Anchored 01igo(dT)2o primer was diluted to 40 pM with PCR grade water, RT master mix was prepared with the composition listed in Table 2 for each reaction. Reverse transcriptase enzyme was added last.Table 2.Component Volume, per sample (pL)5x First-Strand Buffer 4dNTP mix 10 mM 220 mMDTT 2Takara RRI 0.16Oligo dT 40 uM 1drTSO 100 uM 0.4RT quality water 0.44SmartScribe Reverse Transcriptase 2RNA 8Total volume 20

[0059] The next step was PCR 0: pre-amplification of heavy chain genes. Pre¬ amplification of the antibody sequences (e.g., IgG or IgM antibody sequences) from the cDNA samples of the RT step prepares the oligos for subsequent rounds of amplification. Working dilutions of reagents were made using PCR grade water, including TSO F Primer diluted to 10 gM, Reverse Primer was also diluted to 10 gM. PCRO reactions were prepared as shown in Table 3. Components were kept on ice. Polymerase was added last.Table 3.Reagent Volume perreaction (pL)Roche KAPA Biosystems HiFi DNA polymerase,12.52xTSO F primer (10 gM) 0.4Reverse primer (10 gM) 0.4PCR Grade Water 1.7cDNA Template (1:10) 10Total Volume 25

[0060] Samples were covered before brief centrifugation to collect all liquid at the bottom. PCRO reactions were placed in a thermocycler for thermocycling.

[0061] PCRO amplicons can optionally be purified. For example, beads (KAPA HyperPure) were taken from the fridge and allowed to come to room temperature (around 30 minutes). Each sample was transferred to a microcentrifuge tube, after which the sample volume was brought up to 50 pL with dilution by PCR grade water. Beads were vortexed for 5- 10s immediately before drawing an aliquot. 45 pL of the beads (0.9X bead ratio) were added to each 50 pL sample and thoroughly mixed. The bead-sample mixture was incubated for 5 min at room temperature to allow cDNA to bind to the beads. Tubes were placed magnetic racks to capture beads and incubate until liquid was clear. Supernatant was removed and discarded. While the tube remained on the magnetic rack, beads were repeatedly washed by adding enough 80% ethanol such that beads are completely submerged (normally 200 pL). Wash solution was discarded and beads were allowed to air dry while the tube remained on the magnetic rack. Purified products were eluted by adding 20 pL 10 mM Tris-HCl pH 8.0 dropwise onto bead pellet and mixing well. Tubes were again placed on the magnetic rack. Once the solution was clear, the eluate was transferred to a separate tube.

[0062] The next step was PCR1: amplification of heavy chain genes and addition of SBS handles Read 1 and Read 2 (also referred to herein as R1 and R2, respectively). Antibody sequences from PCRO amplicons were amplified using a semi-nested amplification scheme. PCR1 reactions were prepared as shown in Table 4.Table 4.Reagent Volume per reaction(pL)Roche KAPA Biosystems HiFi DNA polymerase, 2x 12.5R2-ISO F primer, 10 uM 0.4 Reverse primer, 10 pM 0.41 (if purified)PCRO product (template)2 (if unpurified)PCR grade water add to reach total of 25Total Volume 25

[0063] Samples were covered before brief centrifugation to collect all liquid at the bottom. PCR1 reactions were placed in a thermocycler for thermocycling. After thermocycling, the PCR1 mixture could optionally be purified in accordance with the discussion with respect to PCR0. After thermocycling, the PCR1 mixture were quantified.

[0064] The next step was PCR2: addition of indexing sequences (e.g., i7 index) and clustering sequences (e.g., P5 and P7 sequences). Working dilutions of forward and reverse primer were prepared using PCR grade water, diluting the Index _fwd primer and Index_rev_00## to 10 pM. Each library included a unique reverse index. PCR2 reactions were prepared in accordance with Table 5. All components were kept on ice. Polymerase was added last.Table 5.Reagent Volume per reaction (gL) Roche KAPA Biosystems HiFi DNA polymerase,25 2xIndex fwd Primer, 10 pM 5 Index_Rev_00## Primer, 10 pM 5 DNA Template 0.5 ng-2 ng PCR Water add to reach total of 50 Total Volume 50

[0065] Sample mixtures were capped and centrifuged briefly to collect all liquid at the bottom before being placed in a thermocycler for thermocycling. The PCR2 step resulted in the final product with reference to FIG. 2, including, among other things, clustering sequences, an index sequence, sequencing handles, the TSO forward oligo, the UTR sequence, and the V(D)J sequence.Example 2 ---- Construction of NGS Library from Sorted Splenocytes

[0066] FIG. 3 shows an example process for constructing an NGS library from sorted cells, for example splenocytes or lymphocytes. In some examples, the sorted cells are rodent cells, for instance mouse or rat cells. Construction of the NGS library was carried outsimilarly to the procedure discussed with respect to Example 1. Certain differences in the protocols are discussed below. For example, the primers used are laid out below in Table 6. Table 6.Reaction Antibody isotype Forward primer Reverse primerRT Any SEQ. ID. No. 1 (drTSO) SEQ. ID. No. 2 Anchored oligo(dT)20)PCR0 Rat IgG SEQ. ID. No. 4 (TSO_F) SEQ. ID. No. 5(RatIGHG CH2 rev outer)Rat IgG SEQ. ID. No. 4 (TSO_F) SEQ. ID. No. 6(RatIGHG CH 1 rev outer)Rat IgM SEQ. ID. No. 4 (TSO_F) SEQ. ID. No. 7(RatIGHM rev outer)PCR1 Rat IgG SEQ. ID. No. 9 (R2-TSO_F-X##, where X## is SEQ. ID. No. 10(RatIGHG_rev_inner_R1) a well ID)Rat IgM SEQ. ID. No. 9 (R2-TSO_F-X##, where X## is SEQ. ID. No. 11(RatIGHM_rev_inner_R1) a well ID)PCR2 Any index_fwd index_rev_00#

[0067] For the RT step, reagents were prepared in accordance with Table 7.Table 7.Component Volume, per sample (pL) Takara 10X lysis buffer 0.8 Takara Recombinant RNAse inhibitor 0.16RT PCR quality water 6.64 Takara 5X First- Strand Buffer 4 Takara 20 mM DTT 2 Takara SMARTScribe Reverse Transcriptase 2 Oligo dT20, 40 μM 2 dNTP, 10 mM 2 TSO primer, 100 μM 0.4 Total Volume 20

[0068] For the PCRO step, reagents were prepared in accordance with Table 8.Table 8.Reagent Volume per reaction (yL) Roche KAPA Biosystems HiFi DNA polymerase, 2x 12.5 R2-TSO_F_## primer, 10 μM 0.4 Reverse primer, 10 μM 0.4 PCRO product (template) 5 PCR grade water 6.7 Total V olume 25

[0069] For the PCR1 step, reagents were prepared in accordance with Table 9. Table 9.Reagent Volume per reaction (pL)Roche KAPA Biosystems HiFi DNA12.5 polymerase, 2xR2-TSO_F_## primer, 10 uM 0.4 Reverse primer, 10 pM 0.4 PCRO product (template) 5 PCR grade water 6.7 Total Volume 25

[0070] PCR1 step 1 additionally adds a barcode sequence to the amplification products.Example 3 — Construction of NGS Library from Human cells

[0071] NGS libraries can be prepared from human cells using similar methods to those discussed with respect to Examples 2 and 3. Table 10 below lists example suitable primer pairings for PCR 0 and PCR 1 steps. It is to be understood that the same primer pairings that were used for the RT and PCR2 steps with reference to Examples 2 and 3 may also be suitably implemented for preparation using human cells.Table 10.Light Chain Reaction Forward Reverse primerType primerKappa PCRO SEQ. ID. No. 4 SEQ. ID. No. 12(TSO_F) (hVK_rev outer 1 )PCRO SEQ ID. No. 4 SEQ. ID. No. 13(TSO F) (hVK_rev_outer2)PCR1 SEQ. ID. No. 9 SEQ. ID. No. 14{R2-TSO f- (h VK rev inner Rl)X##, where X##is a well ID)Lambda PCRO SEQ. ID. No. 4 SEQ. ID. No. 15C I SO F ") (hVL rev outer 1 )PCRO SEQ. ID. No 4 SEQ. ID. No. 16(TSO_F) (hVL_rev_outer2)PCR1 SEQ. ID. No. 9 SEQ. ID. No. 17(R2-TSO_F- (hVL_rev_inner_R1)X##, where X##is a well ID)Example 4 ------ Construction of NGS Library from Mouse Cells

[0072] NGS libraries can be prepared from mouse cells using similar methods to those discussed with respect to Examples 2 and 3. Table 11 below lists example suitable primer pairings for PCR 0 and PCR 1 steps. It is to be understood that the same primer pairings that were used for the RT and PCR2 steps with reference to Examples 2 and 3 may also be suitably implemented for preparation using mouse cells.Table 11.Antibody Reaction Forward Reverse primerIsotype primerMouse IgG PCR0 SEQ. ID. No. 4 SEQ. ID. No. 18(TSO F) (msIgG CHl outer l )PCR1 SEQ. ID. No. 9 SEQ. ID. No. 19(R2-TSO F- (msIgG_CH1_inner1-1)X##, where X##is a well ID)PCR1 SEQ. ID. No. 9 SEQ. ID. No. 20(R2-TSO F- (msIgG_CH1_inner1-1)X##, where X##is a well ID)Example 5 - -- Construction of NGS Library from Rabbit Ceils

[0073] NGS libraries can be prepared from rabbit cells. To amplify VH and VL from rabbit B cells, a template-switching primer was used. In the first step, cells were lysed (e.g., using Takara Lysis Buffer) and reverse transcription carried out using a reverse transcriptase (e.g., SmartScribe, Takara, 639537) with oligo(dT)2o and a TSO adaptor primer (e.g., the adaptor primer having a sequence of SEQ. ID. No. 1). To amplify V-genes, a serni-nested approach was employed. While not being bound to a particular theory, rabbits are believed to preferentially use VH1, the most proximal V gene segment, and have only one gene for IgG. See Becker and Knight (1990) Somatic Diversification of Immunoglobulin Heavy Chain VDJ Genes: Evidence for Somatic Gene Conversion in Rabbits. Cell (63) 967-997. doi: 10.1016 / 0092-8674(90)90502-6.; Kodangattil et al., (2014) The Functional Repertoire of Rabbit Antibodies and Antibody Discovery Via Next-Generation Sequencing. mAbs, 6:3, 628-636. DOI: 10.4161 / mabs.28059; both of which are incorporated by reference in their entirety.

[0074] In the first PCR, the VH was amplified with TSO-F2 (SEQ. ID. No. 9) and a primer located in CH2, Rabbit VH rev outer (SEQ. ID. No. 21). TSO-F2 contains a definedbarcode sequence (represented in the sequence listing by “NNNNNNNN”), which allowed for indexing in subsequent amplifications for next generation sequencing. The conditions for this amplification were: an initial denaturation at 95 °C for 3 min; 18 cycles of denaturation at 98 °C for 20s; annealing at 54 °C for 15s; and extension at 72 °C for 30 s. A final extension was performed at 72 °C for 5 min.

[0075] In the second PCR, TSO-F2 and RabbitVH_rev inner (SEQ. ID. No. 22) were paired to produce the final amplicon. The conditions for this amplification were: an initial denaturation at 95 °C for 3 min; 28 cycles of denaturation at 98 °C for 20s; annealing at 56 °C for 15s; and extension at 72 °C for 30 s. A final extension was performed at 72 °C for 5 min. The distance between VH-outer and VH-inner primers was 154 base pairs,

[0076] The kappal locus may be preferred in rabbits, representing 70% of light chains in the antibody repertoire. See F Ros, N Reichenberger, T Dragicevic, WC van Schooten, R Beulow, and J Platzer, (2005), Sequence analysis of 0.4 megabases of the rabbit germline immunoglobulin kappal light chain locus. Animal Genetics 36(1); 51-57, doi: 10.1111 / j.1365-2052.2004.01221.x, (incorporated by reference in its entirety). A semi-nested approach was again employed to amplify kappa light chains. In the first PCR, TSO-F2 (SEQ. ID. No. 9) and RabbitVK_rev_outer (SEQ. ID. No. 23), which is located at the 3’ end of the constant region, were used. The conditions for the amplification were: an initial denaturation, 95 °C for 3 min; 28 cycles of denaturation at 98 °C for 20s; annealing at 56 °C for 15s; extension at 72 °C for 30 s. A final extension was performed at 72 °C for 5 min. In the second PCR, TSO-F2 (SEQ. ID. No. 9) and RabbitVK rev inner (SEQ. ID. No. 24) were used. The conditions for this amplification were: an initial denaturation at 95 °C for 3 min; 25 cycles of denaturation at 98 °C for 20s; annealing at 56 °C for 15s; and extension at 72 °C for 30 s. A final extension was performed at 72 °C, 5 min. The distance between the VK-outer and VK-inner primers was 70 bp.

[0077] FIGS. 4A and 4B are images taken from a western blot showing representative example rabbit VH and VK amplification, respectively, from single cells. Rabbit splenocytes were incubated with IgG capture beads coated with anti-rb IgG (e.g., Clone 2A9, Southern Biotech) and antigen-tagged (e.g., with AlexaFluor 647). Following a 1.5 hr incubation at 37 °C, antigen positive cells were isolated and lysis buffer was added. VH (toppanel) and VK (lower panel) were amplified as described above. The expected size of VH and VK amplicons were approximately 726 and 650 bp, respectively.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a nucleic acid for antibody sequencing, the method comprising:(a) preamplifying an antibody cDNA sequence to generate a preamplified antibody cDNA sequence by introducing a first forward primer and a first reverse primer to the antibody cDNA sequence, the first forward primer comprising a template switch oligo (TSO) sequence;(b) amplifying the preamplified antibody cDNA sequence of (a) to generate a first amplicon, the amplifying of (b) comprising introducing a second forward primer and a second reverse primer, the second forward primer comprising a TSO sequence and a first priming sequence, the second reverse primer comprising a second priming sequence; and(c) amplifying the first amplicon of (b) to generate a second amplicon, the amplifying of (c) comprising introducing a third forward primer and a third reverse primer, the third forward primer comprising a first portion corresponding to a length of the first priming sequence and a second portion comprising a first clustering sequence, the third reverse primer comprising at least a first portion corresponding to the first priming sequence and a second clustering sequence.

2. The method of claim 1, wherein (a) comprises using a plurality of first reverse primers each having different sequences, and wherein the first forward primer comprises only SEQ. ID. No. 1.

3. The method of claim 1 or claim 2, wherein the first reverse primer binds to the constant region of the antibody cDNA sequence at a first position, wherein the second reverse primer binds to a constant region of the amplified antibody cDNA sequence at a second position, and wherein a 5’ end of the first position is separated from a 5’ end of the second position by a range of from about 49 to about 351 base pairs.

4. The method of claim 3, wherein the 5’ end of the first position is separated from the 5’ end of the second position by about 49, 70, 75, 99, 111, 153, 154, 155, 204, or 351 base pairs.

5. The method of any one of claims 1 to 4, comprising reverse transcribing sample RNA using a reverse transcriptase to generate the antibody cDNA sequence, wherein the reverse transcribing adds the TSO sequence to the antibody cDNA sequence.

6. The method of claim 5, wherein the sample RNA is a rodent RNA sample, a human RNA sample, or a chicken RNA sample.

7. The method of claim 6, wherein the sample RNA is a mouse RNA sample, a rat RNA sample, or a rabbit RNA sample.

8. The method of claim 5, wherein the reverse transcribing also adds a unique molecular identifier (UMI) sequence to the antibody cDNA sequence.

9. The method of any one of claims 1 to 8, wherein the preamplifying of (a) selects for an antibody cDNA oligonucleotide having a sequence corresponding to the TSO forward primer and the first reverse primer.

10. The method of any one of claims 1 to 9, wherein the antibody cDNA sequence comprises an IgG sequence or an IgM sequence.

11. The method of any one of claims I to 10, wherein one of the first clustering sequence or the second clustering sequence comprises a P7 sequence, and wherein the other of the first clustering sequence or the second clustering sequence comprises a P5 sequence.

12. The method of claim 11, wherein the first clustering sequence comprises a P7 sequence and wherein the second clustering sequence comprises a P5 sequence.

13. The method of any one of claims 1 to 12, wherein one of the third forward primer and the third reverse primer comprises an indexing sequence.

14. The method of any one of claims 1 to 13, the antibody cDNA sequence comprising a V(D)J sequence.

15. The method of any one of claims 1 to 14, wherein the first priming sequence and the second priming sequence are next generation (NGS) priming sequences.

16. The method of claim 15, wherein the first priming sequence and the second priming sequence are sequencing-by-synthesis (SBS) priming sequences.

17. The method of claim 16, wherein one of the first priming sequence or the second priming sequence comprises a Read 1 sequence, and wherein the other of the first priming sequence or the second priming sequence comprises a Read 2 sequence.

18. The method of claim 17, wherein the first SBS priming sequence comprises a Read 2 sequence, and wherein the second SBS priming sequence comprises a Read 1 sequence.

19. The method of any one of claims 1 to 18, wherein the first reverse primer comprises one of SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 18, SEQ. ID. No. 21, or SEQ. ID. No. 23.

20. The method of any one of claims 1 to 19, wherein the second reverse primer comprises one of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ID. No. 17, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No. 22, or SEQ. ID. No. 24.

21. The method of any one of claims 1 to 18, wherein the first reverse primer comprises one of SEQ. ID. No. 5 or SEQ. ID. No. 6, and the second reverse primer comprises SEQ. ID. No. 10.

22. The method of any one of claims 1 to 18, wherein the first reverse primer comprises SEQ. ID. No. 7 and the second reverse primer comprises SEQ. ID. No. 11.

23. The method of any one of claims 1 to 18, wherein the first reverse primer comprises SEQ. ID. No. 18 and the second reverse primer comprises SEQ. ID. No. 19 or SEQ. ID. No. 20.

24. The method of any one of claims 1 to 18, wherein the first reverse primer comprises SEQ. ID. No. 21 and the second reverse primer comprises SEQ. ID. No. 22.

25. The method of any one of claims 1 to 18, wherein the first reverse primer comprises SEQ. ID. No. 23 and the second reverse primer comprises SEQ. ID. No. 24.

26. The method of any one of claims 1 to 25, comprising obtaining an RNA sample from a single cell.

27. The method of any one of claims 1 to 25, comprising obtaining an RNA sample from less than 1000 cells,28. The method of any one of claims 1 to 25, comprising obtaining an RNA sample from a bulk cell sample.

29. A method of determining an antibody sequence, the method comprising: completing the method of any one of claims 1 to 28 to produce a sequencing product; andsequencing the sequencing product.

30. The method of claim 29, wherein the sequencing comprises conducting next generation sequencing (NGS).

31. The method of claim 30, wherein the sequencing comprises conducting sequencing by synthesis (SBS).

32. An amplicon prepared by the method of any one of claims 1 to 28.

33. A kit for preparing a nucleic acid for antibody sequencing, the kit comprising:a first forward primer, the first forward primer comprising a template switch oligo (TSO) sequence;a first reverse primer;a second forward primer comprising a TSO sequence and a first priming sequence;a second reverse primer comprising a second priming sequence; a third forward primer comprising a first portion corresponding to a length of the first priming sequence and a second portion comprising a first clustering sequence; anda third reverse primer comprising at least a first portion corresponding to the first priming sequence and a second clustering sequence.

34. The kit of claim 33, comprising a plurality of first reverse primers having different sequences, and wherein the first forward primer comprises only the TSO sequence.

35. The kit of claim 33 or claim 34, wherein the first priming sequence and the second priming sequence are next generation (NGS) priming sequences.

36. The kit of claim 35, wherein the first priming sequence and the second priming sequence are sequencing-by-synthesis (SBS) priming sequences.

37. The kit of claim 36, wherein one of the first priming sequence or the second priming sequence comprises a Read 1 sequence, and wherein the other of the first priming sequence or the second priming sequence comprises a Read 2 sequence.

38. The kit of claim 37, wherein the first priming sequence comprises a Read 2 sequence, and wherein the second priming sequence comprises a Read 1 sequence.

39. The kit of any one of claims 33 to 38, wherein one of the third forward primer and the third reverse primer comprises an indexing sequence.

40. The kit of any one of claims 33 to 39, wherein one of the first clustering sequence or the second clustering sequence comprises a P7 sequence, and wherein the other of the first clustering sequence or the second clustering sequence comprises a P5 sequence.

41. The kit of claim 40, wherein the first clustering sequence comprises a P7 sequence and wherein the second clustering sequence comprises a P5 sequence.

42. The kit of any one of claims 33 to, wherein the first reverse primer and the second reverse primer comprise sequences corresponding to portions of a constant region of a target cDNA oligonucleotide.

43. The kit of any one of claims 33 to 42, wherein the first reverse primer comprises one of SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 18, SEQ. ID. No. 21, or SEQ. ID. No. 23.

44. The kit of any one of claims 33 to 43, where the second reverse primer comprises one of SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 14, SEQ. ID. No. 17, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No. 22, or SEQ. ID. No. 24.

45. The kit of any one of claims 33 to 42, wherein the first reverse primer comprises one of SEQ. ID. No. 5 or SEQ. ID. No. 6, and the second reverse primer comprises SEQ. ID. No. 10.

46. The kit of any one of claims 33 to 42, wherein the first reverse primer comprises SEQ. ID. No. 7 and the second reverse primer comprises SEQ. ID. No. 11.

47. The kit of any one of claims 33 to 42, wherein the first reverse primer comprises SEQ. ID. No. 18 and the second reverse primer comprises SEQ. ID. No. 19 or SEQ. ID. No.20.

48. The kit of any one of claims 33 to 42, wherein the first reverse primer comprises SEQ. ID. No. 21 and the second reverse primer comprises SEQ. ID. No. 22.

49. The kit of any one of claims 33 to 42, w’herein the first reverse primer comprises SEQ. ID. No. 23 and the second reverse primer comprises SEQ. ID. No. 24.

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