Methods and compositions for generating cell source-identifiable nucleic acid collections

Abstract

A method is provided for preparing a source-identifiable nucleic acid collection from multiple sources, such as cells or cell nuclei, using a combinatorial indexing methodology. In general, the method includes providing a first set of cell source subportions, each subportion including a plurality of cell sources from an initial plurality of cell sources. A template-switching mediated reaction is then used to generate a first identifier-tagged nucleic acid in the plurality of cell sources in each subportion of the first set, using a template switch oligonucleotide including a first identifier, and the first identifier of the template switch oligonucleotide used in different subportions of the first set is the same within a given subportion but different between different subportions. The cell sources of the subportions are then pooled to generate a first cell source pool including the first identifier-tagged nucleic acid, and the first pool is then allocated to a second set of subportions, each including a plurality of cell sources having the first identifier-tagged nucleic acid. Then, cell-source-identifiable nucleic acids are produced from the multiple cell sources in each subportion of the second set that include both the first identifier and the second identifier (the second identifier of each subportion of the second set is the same within a given subportion, but different between different subportions) to prepare a multiple cell-source-identifiable nucleic acid collection from the initial multiple cell sources. The nucleic acids of each cell-source-identifiable nucleic acid collection include a unique combination of the first and second identifiers that identify the cell source of the nucleic acid. Also provided are kits, compositions, and devices for use in carrying out embodiments of the methods described herein, for example.

Description

[Background technology]

[0001] The development of next-generation sequencing (NGS) technologies has enabled the rapid extraction of valuable genomic and transcriptomic information from generated nucleic acid libraries.

[0002] High-throughput NGS technologies, such as sequencing platforms provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ Sequencing Systems), Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ Sequencing Systems), Pacific Biosciences (e.g., PACBIO RS II Sequel Sequencing System), Life Technologies™ (e.g., SOLiD™ Sequencing System), Roche (e.g., 454 GS FLX+ and / or GS Junior Sequencing Systems), and others, allow for the sequencing of nucleic acid molecules more quickly and cheaply than previously used Sanger sequencing, and thus these technologies have revolutionized biotechnology and biomedical research. Moreover, as these technologies mature and become more user-friendly, their presence in clinical applications continues to increase.

[0003] These powerful sequencing techniques place a particular emphasis on library preparation: well-prepared and efficiently generated reverse-transcribed complementary DNA (cDNA) libraries can be analyzed using NGS techniques for a diverse range of purposes.

[0004] Current NGS workflows allow for sequencing of libraries prepared from samples obtained from bulk cell populations or single cells. Sequencing of bulk cell populations does not allow for analysis of genomic and / or transcriptomic changes at single-cell resolution and may mask the underlying heterogeneity of different cell types in bulk populations. When nucleic acid libraries are prepared from individual cells, NGS techniques allow for analysis of genomic and / or transcriptomic changes at single-cell resolution. Single-cell sequencing offers many advantages over bulk cell sequencing, but single-cell sequencing requires the ability to trace a given nucleic acid back to its original source.

[0005] Although sample barcoding techniques have been developed to address this requirement, there is still an upper limit on the number of different cells that can be realistically processed in a given experiment. In some cases, the number of cells that are desired to be processed in a single experiment exceeds the number that can be easily processed using current protocols, for example, if all of the cells are ultimately pooled in a single sequencing-ready library composition. For example, with respect to processing of human adaptive immune repertoire samples, single-cell analysis allows pairing chain information (e.g., alpha / beta / gamma / delta pairing for TCR and heavy / light pairing for BCR). However, the number of cells that can be examined in a given experiment may be limited, for example, up to 10,000 different cells, or require specialized instrumentation. Summary of the Invention

[0006] In such cases, the inventors recognize that what is needed is an approach that allows for the analysis of more than 10,000 cells in parallel. Ideally, what is needed is a method that allows for the analysis of more than 10,000 cells in parallel, and further, where such a method does not require specialized equipment beyond the instrumentation known in the current state of the art. For example, what is needed in the art is an approach that allows for more than 100,000 cells, such as up to 1 million single cells, or more than 1 million cells, to be analyzed in parallel in a single experiment (e.g., providing paired chain information for TCR or BCR). In addition, the inventors also recognize that what is needed in the art is a method for performing single cell experiments on multiple samples of single cells, where the number of single cells in each sample may be small, but the number of independent samples is large, and therefore the number of sets of cells that need to be analyzed is large. For example, what is needed in the art is an approach that allows for the analysis of, for example, 100 or more samples (each sample containing 1,000 or more cells), such that the total number of cells analyzed is 100,000 or more. Therefore, there is a continuing need for improved single-cell sequencing techniques that can accommodate the processing of large numbers of cells in a given experiment.

[0007] The present invention fulfills these and other needs in the art by providing a combinatorial indexing approach for uniquely identifying nucleic acids produced from the same cellular source. The combinatorial indexing approach used by the present invention embodiments described herein represents a substantial improvement over the art by providing this unique identification without the need for each cell in the assayed population to be individually present in a container separated from other cells to be assayed. The provided combinatorial approach allows for practical analysis of large numbers of single cells, e.g., 10,000 or more single cells, 100,000 or more single cells, or 1 million or more single cells. The provided combinatorial approach is equally applicable to its components, e.g., nuclei, using the same workflow. For example, in this same workflow, sequencing-ready libraries of nucleic acids from, e.g., 100,000 or more single cells or single nuclei, are pooled and sequenced together, and the resulting sequence information of each read can be traced back to its original cellular source. Furthermore, the methods for combinatorial analysis provided herein allow for the practical analysis of many independent cell samples, such as, for example, the analysis of 100 or more samples (each sample containing 1,000 or more cells) such that the total number of cells analyzed is 100,000 or more.

[0008] A method is provided for preparing a multiple cell source identifiable nucleic acid collection derived from an initial multiple cell sources. An embodiment of this method includes providing a first set of cell source subportions, each subportion including multiple cell sources from the initial multiple cell sources. A template-switching mediated reaction is then used to generate a first identifier-tagged nucleic acid in the multiple cell sources of each subportion of the first set, using a template switch oligonucleotide including a first identifier (also referred to herein as a first index or a first cell barcode), where the first identifier of the template switch oligonucleotide used in different subportions of the first set is identical within a given subportion but different between different subportions. The cell sources of the subportions are then pooled to generate a first cell source pool including the first identifier-tagged nucleic acid, and the first pool is then allocated into a second set of subportions, each including multiple cell sources having the first identifier-tagged nucleic acid. Then, cell-source-identifiable nucleic acids are produced from the plurality of cell sources in each subportion of the second set, the nucleic acids comprising both the first identifier and the second identifier, and the second identifier of each subportion of the second set is identical within a given subportion but different between different subportions. This method provides a plurality of cell-source-identifiable nucleic acid collections from the initial plurality of cell sources. The nucleic acids of each cell-source-identifiable nucleic acid collection comprise a unique combination of the first and second identifiers that identify the cell source of the nucleic acid.

[0009] In other embodiments, additional rounds of pooling and redistribution into new subportions can be used, as desired, to add additional rounds of indexes (i.e., identifiers) to the nucleic acid collection. Additional rounds of indexing allow the methodology to analyze more individual cells or cellular components, such as nuclei. In general, the total number of cells examined should be such that the total number of unique combinations and orientations of nucleotide sequence identifiers (e.g., barcodes, indexes, tags, or any type of molecular identifier) ​​is greater than the number of unique cells that the researcher wishes to interrogate.

[0010] Also provided are kits, compositions, and devices, eg, for use in carrying out embodiments of the methods described herein. [Brief description of the drawings]

[0011] [Figure 1A] 1 illustrates a workflow according to one embodiment of the present invention. [Figure 1B] 1 illustrates a workflow according to one embodiment of the present invention. [Figure 1C] 1 illustrates a workflow according to one embodiment of the present invention. [Figure 1D] 1 illustrates a workflow according to one embodiment of the present invention. [Diagram 2] 1 provides a schematic diagram illustrating one embodiment of the present invention. [Diagram 3] 1 provides a schematic diagram illustrating one embodiment of the present invention. [Figure 4] 1 provides a schematic diagram illustrating one embodiment of the present invention. [Diagram 5] 1 provides a flow chart outlining one embodiment of the method of the present invention. [Figure 6] 6 provides a schematic diagram showing the structure of an NGS library product generated using an embodiment of the invention as described in Example 2. More specifically, T cell receptor genes are specifically targeted for analysis. As shown in FIG. 6, lead 1 provides the sequence of the targeted TCR gene. Lead 2 also provides the sequence of the T cell receptor gene along with the first two index sequences, namely index 2 (IN2, second identifier) ​​from the second indexing step and index 1 (IN1, first identifier) ​​from the first indexing step. Index 3 (third identifier) ​​is shown as provided by a combination of the i7 and i5 indexes added by PCR in the third indexing step. [Figure 7]A schematic illustrating one embodiment of the present invention is provided, specifically a method for preparing a multi-cell source identifiable nucleic acid collection derived from an initial multi-cell source, which uses three independent rounds of nucleic acid indexing. [Figure 8A] FIG. 1 illustrates a two-round barcoding protocol for TCR sequencing (as described in Example 4 below), according to an embodiment of the invention. [Figure 8B] Illustrated is the experimental design of an 8×8 split pool containing 1,000 cells for two rounds of TCR barcoding. [Figure 8C] Illustrates BioAnalyzer results from the TCRb library for two rounds of TCR barcoding. [Figure 8D] Illustrates L-plot analysis for two rounds of TCR barcoding. [Figure 9] FIG. 1 illustrates two rounds of barcoding for TCR sequencing, with the second round of barcoding split across PCR1 and PCR2, according to an embodiment of the invention. [Figure 10A] FIG. 1 illustrates three rounds of barcoding for TCR sequencing (as described in Example 5 below) with the third round of barcoding split across PCR1 and PCR2, according to an embodiment of the invention. [Figure 10B] FIG. 1 illustrates Bioanalyzer tracking of a TCR library generated as described in Example 5 for three rounds of TCR barcoding. [Figure 10C] 13 is a table showing read counts, mapping rates, and clonotypes detected in the libraries for three rounds of TCR barcoding. [Figure 11] FIG. 1 illustrates an alternative three-round barcoding strategy for TCR sequencing, according to an embodiment of the invention. [Figure 12A]FIG. 1 illustrates two rounds of barcoding for combined targeted sequencing and 5′ differential expression (5′DE) (as described in Example 6 below) according to an embodiment of the invention. [Figure 12B] 12A shows the products and library structures generated using the protocol illustrated in FIG. 12A. [Figure 12C] A knee plot is provided that is used to determine the number of detected cells that pass the quality metric. In this case, cells with >10,000 reads / cell based on data demultiplexing using BC1, BC2a, BC2b (i7) and i5 with Cogent AP software. 1310 cells had >10,000 reads and were used for downstream analysis. [Figure 12D] Average mapping statistics for K562 and 3T3 cells based on mapping to either human hg38 (K562) or mouse mm10 (3T3) are provided. The percentages of intergenic, intronic, exonic, multi-mapped, unmapped, and trimmed reads were calculated. [Figure 12E] The number of genes detected in either K562 or 3T3 cells as a function of reads per cell is provided. [Figure 12F] L-plot analysis is provided. All K562, 3T3 and mixed cell sequencing reads were mapped to both human (hg38) and mouse (mm10) genomes and plotted based on the number of mapped reads per cell mapped to each genome. [Figure 13A] 1 shows the product and library structure of a TCR library prepared after two rounds of split-pool barcoding, as described in Example 7. [Figure 13B] For TCR analysis, full length cDNA prepared with 10 ng of PBMC RNA is shown. [Figure 13C] For TCR analysis, TCRa, TCRb and TCRa+b libraries prepared from full-length cDNA are shown. [Figure 14]FIG. 1 illustrates a three-round barcoding protocol for combined targeted sequencing and 5′ differential expression (5′DE) (as described in Example 8 below) according to an embodiment of the invention. [Figure 15] 15 illustrates libraries and products generated according to various embodiments of the present invention. Figure 15, panel A, shows 5'DE library generation as described in Example 8. Figure 15, panel B, shows TCR library preparation as described in Example 8. [Figure 16] 1 provides the results of a TSO analysis as described in Example 9. [Figure 17] 13 provides knee plots of single cells after demultiplexing, as described in Example 10. [Figure 18] 1 provides an L-plot of a human-mouse cell mixture prepared according to an embodiment of the present invention, as described in Example 10. [Figure 19] 13 provides an L-plot of human-mouse cell mixtures processed for combinatorial indexing using increasing concentrations of PFA and digitonin as described in Example 11.

[0012] definition 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 shares some complementarity. Whether a primer specifically hybridizes to a target nucleic acid depends on the degree of complementarity between the polymer and the target nucleic acid and the temperature at which hybridization occurs (the melting temperature (T) of the primer). MThe melting temperature is determined by factors such as 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 can be experimentally determined or predicted using the following formula: Tm=81.5+16.6(log10[Na+])+0.41(fraction G+C)-(60 / N), where N is the chain length and [Na+] is less than 1M. See 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 different parameters may be used to predict the Tm of a primer / target duplex in response to different hybridization conditions. Approaches for achieving specific nucleic acid hybridization can be found, 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 acid probe assays," Elsevier (1993).

[0013] The terms "complementary" and "complementarity" as used herein refer to a nucleotide sequence that non-covalently base pairs to all or a region of a target nucleic acid (e.g., a region of a product nucleic acid). In canonical Watson-Crick base pairing, adenine (A) base pairs with thymine (T) as does guanine (G) with cytosine (C) in DNA. 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 vice versa. Typically, "complementary" refers to a nucleotide sequence that is at least partially complementary. The term "complementary" can also encompass a duplex that is fully complementary, such that every nucleotide in one strand is complementary to every nucleotide in the other strand at the corresponding position. In certain cases, a nucleotide sequence can be partially complementary to a target, where every nucleotide is not complementary to every nucleotide in the target nucleic acid at every corresponding position. For example, a primer may be perfectly (i.e., 100%) complementary to the target nucleic acid, or the primer and target nucleic acid may share some degree of less than perfect complementarity (e.g., 70%, 75%, 85%, 90%, 95%, 99%).

[0014] The percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence for optimal alignment). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100). If a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, then the molecules are identical at that position. For a non-limiting example of such a mathematical algorithm, see 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 utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one embodiment, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., wordlength=5 or wordlength=20).

[0015] As used herein, an "oligonucleotide" is a single-stranded multimer of nucleotides of 2 to 500 nucleotides, e.g., 2 to 200 nucleotides. Oligonucleotides can be synthetic or enzymatically produced, and in some embodiments, can be 10 to 50 nucleotides in length. Oligonucleotides can contain ribonucleotide monomers (i.e., oligoribonucleotides or "RNA oligonucleotides") or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides or "DNA oligonucleotides"). In some cases, oligonucleotides can contain a mixture of ribonucleotides and deoxyribonucleotides. In some cases, oligonucleotides may contain modified, i.e., non-natural nucleotides or modifications including, for example, LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, etc., linkage modifications (e.g., phosphorothioates, 3'-3' and 5'-5' reverse linkages), 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 a desired function to the oligonucleotide. Oligonucleotides may be, for example, 10-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 80-100, 100-150, or 150-200, up to 500 nucleotides or more in length.

[0016] A "domain," when used in reference to a nucleic acid, refers to a stretch or length of a nucleic acid consisting of multiple nucleotides, which stretch or length provides a defined function to the nucleic acid. Examples of domains include barcode domains (such as source barcode domains), primer binding domains, hybridization 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, e.g., when an immune receptor chain domain / region is described, it includes an 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, e.g., 5 to 50 nt, e.g., 5 to 30 nt. An amplification primer binding domain is a domain that is configured to bind to an amplification primer via hybridization.

[0017] As used herein, the phrase "derived from" describes a composition resulting from a process in which a first component (e.g., a first nucleic acid molecule), or information from that first component, is used to isolate, derive, or construct a different second component (e.g., a second nucleic acid molecule that differs in structure, sequence, or characteristics from the first nucleic acid molecule from which it is derived). For example, a cDNA molecule is derived from the corresponding mRNA found in a cell. Similarly, a DNA library is derived from total RNA collected from a cell or cell population. Also, for example, a cDNA library can be derived from mRNA collected from a cell or cell population.

[0018] As used herein, the term "barcode" most broadly describes a short sequence, e.g., 6-12 nucleotides, which, when added to a larger polynucleotide, serves to tag the larger polynucleotide, thereby providing a means to count or distinguish individual nucleic acids within a larger pool of nucleic acids. As used herein, and as will be recognized by those skilled in the art, a wide variety of barcodes and barcoding strategies are widely available and described in the prior art, all of which are used in the invention described herein. As used herein, the term "barcode" or "index" may be used interchangeably with the terms tag, identifier tag, cell barcode, cell barcode sequence, sample barcode and sample barcode sequence, well barcode, source barcode sequence, identifier, molecular identifier, and other similar and equivalent expressions and technologies. The term "unique molecular identifier" or "UMI" also refers to randomers of various lengths, and is also encompassed by the broader definition of "barcode" as used herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] A method for preparing a multiple cell source identifiable nucleic acid collection from an initial multiple cell sources is provided. An embodiment of this method includes providing a first set of cell source subportions, each subportion including multiple cell sources from the initial multiple cell sources. A template-switching mediated reaction is then used to generate a first identifier-tagged nucleic acid in the multiple cell sources of each subportion of the first set, using a template switch oligonucleotide including a first identifier, and the first identifier of the template switch oligonucleotide used in different subportions of the first set is the same within a given subportion but different between different subportions. The cell sources of the subportions are then pooled to generate a first cell source pool including the first identifier-tagged nucleic acid, and the first pool is then allocated to a second set of subportions, each including multiple cell sources having the first identifier-tagged nucleic acid. Then, cell-source-identifiable nucleic acids are produced from the multiple cell sources in each subportion of the second set that includes both the first identifier and the second identifier (the second identifier of each subportion of the second set is the same within a given subportion but different between different subportions) to prepare a multiple cell-source-identifiable nucleic acid collection from the initial multiple cell sources. The nucleic acids of each cell-source-identifiable nucleic acid collection include a unique combination of the first and second identifiers that identify the cell source of the nucleic acid. One method of the present invention is illustrated in the flow chart provided in FIG. 5.

[0020] In other embodiments, additional rounds of pooling and redistribution into new subportions can be used as desired to add additional rounds of index to the nucleic acid collection. The number of rounds, and the total number of individual cell source subportions in each round, are selected to optimize the method for any particular application of the methodology, i.e., to suit the user's objectives and reflect the total number of cells (i.e., total number of cell sources) that are optimally examined.

[0021] Generally, the total number of cells examined is such that the total number of possible unique combinations and orientations of nucleotide sequence identifiers (e.g., barcodes, tags, or any type of molecular identifier) ​​added in the first and second addition steps (and optional further additions) is significantly greater than the number of unique cells the researcher wishes to examine, such that each cell is likely to obtain a unique combination of nucleotide sequence identifiers and thus be assigned to an individual cell source. As an example, if the number of identifiers is 10 times the number of cell sources, a doublet rate of approximately 5% is achieved. By increasing or decreasing the ratio of identifiers to cell sources, the doublet rate can be increased or decreased as desired.

[0022] Before the invention is described in more detail, it is to be understood that the invention is not limited to the particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing only particular embodiments, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0023] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is to be understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within this stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specific excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0024] A range is provided herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the number that is close to or approximately the unrecited number may be a number that, in the context provided, provides substantial equivalence to the specifically recited number.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but representative exemplary methods and materials are described below.

[0026] 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 to disclose and describe 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 invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0027] It should be 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 should be further noted that the claims may be designed to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0028] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the various other embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0029] Although the apparatus and methods have been or will be described in functional descriptions for grammatical fluidity, it is expressly understood that the claims should not be construed as necessarily limited in any way by the syntax of "means" or "step" limitations unless expressly recited under 35 U.S.C. 112, but should be accorded the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of judicial equivalents, and that the claims, when expressly recited under 35 U.S.C. 112, should be accorded the full statutory equivalents under 35 U.S.C. 112.

[0030] method As summarized above and illustrated diagrammatically in the figures, the present specification provides methods for preparing a multi-cell source identifiable nucleic acid collection from an initial multi-cell source using combinatorial indexing techniques. These methods described herein provide advantages over existing cell combinatorial indexing protocols, as recognized by those skilled in the art. For example, in the methods described herein, full length mRNA transcripts can be combinatorially indexed and assayed without applying long read sequencing techniques. Furthermore, 5' end sequences, such as those in immune cell receptors, can be specifically targeted for analysis.

[0031] cell source As used herein, the phrase "cellular source" refers to a cell or any component thereof that contains nucleic acid. When a cellular component is used, the component is referred to as a "nucleic acid providing component." In some cases, the cellular source can be either a cell or a cell nucleus (wherein the term "nucleus" is used in its conventional sense to refer to the membrane-bound organelle that contains the chromosomes of a cell).

[0032] The initial cell source from which cell source-identifiable nucleic acids are produced according to embodiments of the present invention may be varied and is not particularly limited. Cell samples from which cell sources may be obtained may come from a variety of sources, including, but not limited to, for example, cell tissue, biopsy, blood sample, cell culture, and the like. Furthermore, cell samples may come from specific organs, tissues, tumors, neoplasms, and the like. Furthermore, cells from any population may be the source of cell sources used in the subject methods, such as populations of prokaryotes or eukaryotes, including bacteria or yeast. In some cases, the cell source utilized in the subject methods 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, and the like. In some cases, the mammalian cell sample may be a mammalian blood sample, including, but not limited to, for example, a rodent (e.g., mouse or rat) blood sample, a non-human primate blood sample, a human blood sample, and the like.

[0033] When using cells from an organism or cell culture, it may be preferable to use cells of a specific cell type, such as immune system cells, neural cells, cardiac cells, tumor cells, or any other cell type. When using immune system cells, it may be preferable to further narrow the cell type used in the combined indexing analysis. For example, it may be preferable to limit the analysis to only B cells or T cells. When using cells from whole blood for analysis, it may be preferable to use peripheral blood mononuclear cells (PBMCs).

[0034] If the cell source is a cell nucleus, the nucleus may be obtained from the initial cell using any convenient nucleus isolation protocol. If the cell source is a cell and the cell has not been first isolated, for example, the cell is part of a tissue, the cell may be obtained from the initial cell sample, for example, using any convenient cell isolation protocol.

[0035] The number of cells or cell nuclei in the initial multiple cell source is not particularly limited or restricted to either a minimum number of cells or nuclei requirement or an upper limit of the maximum number of cells or nuclei that can be analyzed, given that sufficient diversity is created by the multiple rounds of indexing used. For example, two, three, or more rounds of indexing may be used in the methods of the invention. The number of cells or cell nuclei in the initial multiple cell source may vary, but in some cases the number of initial multiple cell sources ranges from 2 to 10,000,000, e.g., 1,000 to 1,000,000, including 10,000 to 100,000, and in some cases the number of initial cell sources in the multiple cell source is 100,000 or more, such as 1,000,000 or more. In some embodiments, the initial multiple cell source may include any number of different independent cell samples, e.g., 1 to 100 independent samples or more, e.g., 100 to 1,000 independent samples or more.

[0036] As used herein, the expression "cell-origin identifiable" means that the source of the nucleic acids of a given collection, e.g., a single cell or a single nucleus, can be determined so that the nucleic acids of a given nucleic acid population generated from the same cell source can be traced back to the same or common origin source. In other words, the nucleic acids of a given cell-origin identifiable collection are composed of nucleic acid populations that can be determined to originate from the same source, e.g., cells or nuclei. The method of the present invention provides for the preparation of cell-origin identifiable nucleic acid collections from an initial plurality of cell sources, where each prepared collection can be traced back to a different cell source among the plurality of cell sources. In other words, the method of the present invention allows for the preparation of several nucleic acid collections from several initial cell sources, e.g., cells or nuclei, where each prepared collection can be traced back to its own unique cell source among the initial plurality of cell sources, i.e., the nucleic acids can be determined to originate from its own unique cell source among the initial plurality of cell sources. The cell source-identifiable nucleic acid identifier components, e.g., the first and second identifiers as described in more detail below, allow retroactive identification of the source of a given nucleic acid collection, e.g., cells or nuclei, and thus collectively serve as a source barcode for the nucleic acids of the collection. Furthermore, as will be appreciated by those skilled in the art, the cell source used by the present method is not limited to including, for example, individual cells from a cell culture. The cell source used by the present method can be cell sources from different populations or different samples, e.g., different patients, different cell cultures, different treatment groups, breeding populations, or different plants from different bacterial species, etc.

[0037] Production of a first set of subportions The method aspect of the present embodiment includes providing a first set of cell source subportions, each subportion including a plurality of cell sources of the initial plurality of cell sources. In this step, the initial plurality of cell sources is divided into several subportions, which collectively constitute the first set, each subportion including a plurality of different cell sources. In other words, a plurality of subportions, each consisting of a plurality of cell sources, are generated from the initial plurality of cell sources. If desired, the subportions may be in a container or vessel isolated from each other by a solid barrier, such as, for example, a well of a multi-well plate. Alternatively, the subportions may be contained in droplets, as known in the art, e.g., the droplets are distinct from any other droplets in the droplet collection. The number of subportions that constitute the first set may vary, but in some cases the number ranges from 2 to 25,000 subportions, e.g., 96 to 10,000 subportions, e.g., 96 to 384 subportions. As noted above, each sub-portion of the first set is made up of, i.e., includes, multiple cell sources. In some embodiments in which multiple cell samples are analyzed, each independent sample is considered to be a sub-portion of the initial cell source collection. The number of cell sources that make up a given sub-portion of the first set can vary, but in some cases, the number ranges from 1 to 10,000, such as 10 to 1,000, including 100 to 1,000.

[0038] For example, as described above and below, the subportions used in the methods of the invention may take a variety of different forms. The subportions may take the form of any suitable reaction vessel, including, but not limited to, for example, tubes, wells of a multi-well plate, and the like. In some cases, the subportions are wells of a multi-well device (e.g., a multi-well plate or a multi-well chip or droplet, etc.). In some cases, the components required for a particular reaction step may be placed in the reaction vessel prior to the addition of other reagents, e.g., the reaction vessel may be pre-prepared with one or more components of the reaction. For example, the reaction vessel may be pre-prepared with one or more oligonucleotides, including cases where such oligonucleotides are placed in the reaction vessel in hydrated (e.g., solution or droplet) or dehydrated (e.g., dried, lyophilized) form. Dehydrated reaction components, e.g., lyophilized oligonucleotides and / or enzymes, may be rehydrated in the reaction vessel prior to use or may be rehydrated during the addition of other reaction components or cell sources. There may be a variety of reaction vessels that may serve as subportions to which the reaction mixture and its components may be added and in which the reactions of the subject methods may be carried out. Useful reaction vessels include, but are not limited to, tubes (e.g., single tubes, multi-tube strips, etc.), wells (e.g., multiwell plates (e.g., 96-well plates, 384-well plates, or plates having any number of wells, such as 2000, 4000, 6000, or 10,000 or more). Multiwell plates can be freestanding or part of a chip and / or device, for example, as described in more detail below. For example, 96-well plates, 384-well plates, or plates having any number of wells, such as 2000, 4000, 6000, or 10,000 or more. Multiwell plates can be part of a chip and / or device. The present disclosure is not limited to the number of wells in a multiwell plate. In various embodiments, the total number of wells on the plate is between 100 and 200,000, or between 5,000 and 10,000. In other embodiments, the plate comprises smaller chips, each containing 5,000 to 20,000 wells.For example, a square chip may contain 125×125 nanowells with a diameter of 0.1 mm. The wells (e.g., nanowells) in a multiwell plate may be fabricated in 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 5 mm deep or more. In some cases, the wells may have a depth of 5 mm or less, including, for example, but not limited to, 4 mm or less, 3 mm or less, 2 mm or less, 1 mm or less. In various embodiments, each nanowell has an aspect ratio (ratio of depth to width) of 1 to 6 or more. In one embodiment, each nanowell has an aspect ratio of 1:6. The cross-sectional area may be circular, elliptical, oval, conical, rectangular, triangular, polyhedral, or any other shape. The cross-sectional area at any given depth of the wells may also vary in size and shape. In certain embodiments, the wells have a volume of 0.1 nL to 1 μL. The nanowells may have a volume of 1 μL or less, for example 500 nL or less. The volume may be 200 nL or less, such as 100 nL or less. In one embodiment, the volume of the nanowell is 100 nL. If necessary, the nanowells may be fabricated to increase the surface area to volume ratio, thereby facilitating heat transfer through the unit, which may reduce the ramp time of thermal cycling. The cavity of each well (e.g., nanowell) may take on a variety of configurations. For example, the cavity within the well may be divided by linear or curved walls to form separate but adjacent compartments, or may be divided by circular walls to form inner and outer annular compartments. In some embodiments, a multiwell plate is used, for example in the form of an array of addressable nanowells. An example of such a multiwell plate is one that is part of the ICELL8® Single-Cell MSND System (Takara Bio USA). Further details of the ICELL8® MSND system can be found in U.S. Pat. Nos. 7,833,709 and 8,252,581, and published U.S. Patent Application Publication Nos. 2015 / 0362420 and 2016 / 0245813, the disclosures of which are incorporated herein by reference.

[0039] Generation of a first identifier-tagged nucleic acid Following generation of a first set of subportions, for example as described above, the method of the present invention includes generating a first identifier-tagged nucleic acid in a plurality of cell sources for each subportion of the first set using a template switching-mediated reaction. The first identifier-tagged nucleic acid generated or produced in this step is a nucleic acid that includes a first identifier domain or region. A given first identifier domain can vary in length without limitation, for example, in some cases ranging from 4 to 20 nucleotides (nt), e.g., 8 to 12 nt, and in some embodiments will have a sequence that is distinguishable from the sequence of other first identifier domains used in a given method. In some embodiments, the first identifier domain will also be different from a second or additional identifier domain used in a subsequent step of the method. Depending on the protocol used to generate the first identifier-tagged nucleic acid, the first identifier can be present on the first identifier-tagged nucleic acid at a position that is close to the end or terminus of the tagged nucleic acid.

[0040] In yet other embodiments, it is possible that the first barcode (i.e., the first identifier domain sequence) and the second or subsequent barcode (i.e., the second identifier domain sequence) are identical and can still be used to generate a cell-origin-identifiable nucleic acid collection. That is, the fact that the barcodes are added in different rounds allows the barcodes to be distinguished by the order of addition, without requiring that the barcodes in each round be different from the barcodes in another barcoding round.

[0041] As described above, the first identifier-tagged nucleic acid is generated in a plurality of cellular sources, which means that the first identifier-tagged nucleic acid is generated inside an intact but permeabilized cellular source. Thus, when the cellular source is a cell, the first identifier-tagged nucleic acid is generated inside the cell. Similarly, when the cellular source is a nucleus, the first identifier-tagged nucleic acid is produced inside the nucleus. The cellular source may be permeabilized to allow the reagents used in this step to access the initial template nucleic acid of the cellular source. Any convenient protocol for permeabilizing the cellular source, e.g., the cell or nucleus, may be used. As used herein, the term "permeabilize" refers to making a membrane, e.g., the cell membrane or nuclear envelope, permeable to the reagents used in the template switching-mediated reaction, e.g., the template switch oligonucleotide, reverse transcriptase, first strand cDNA primer, etc. As used herein, the term "permeability" refers to the ability of enzymes, oligonucleotides (e.g., template switch oligonucleotides or primers), etc., or other materials to pass through a lipid bilayer membrane, such as the cell membrane or nuclear envelope, which is the membrane surrounding the nucleus. The term "permeability" may be a relative term indicating the permeability of a particular reagent (e.g., of a particular size) relative to other reagents. In the embodiments described herein, the cellular source, e.g., cell or nucleus, remains structurally intact during permeabilization. In the embodiments described herein, permeabilization may be performed by contacting the cellular source with a chemical agent capable of porosifying the cell and / or organelle membrane. In some cases, the chemical agent is a detergent, and permeabilization may be performed by contacting the cellular source with a buffer containing one or more detergents. The term "detergent" as used herein refers to an amphiphilic (partially hydrophilic / polar and partially hydrophobic / non-polar) surfactant or a mixture of amphiphilic surfactants.Detergents can be broadly classified according to the charge of their polar moiety as "anionic" (negatively charged; including, but not limited to, bile acids such as alkylbenzenesulfonates and deoxycholic acid), "cationic" (positively charged; including, but not limited to, quaternary ammonium and pyridinium-based detergents), "nonionic" (uncharged; including, but not limited to, polyoxyethylene / PEG-based detergents such as Tween and Triton, and glycoside-based detergents such as HEGA and MEGA), and "zwitterionic" (uncharged due to an equal number of positive and negative charges on the detergent molecule; including, but not limited to, CHAPS and amidosulfobetaine-type detergents). In some embodiments, suitable detergents for permeabilizing cell sources include, but are not limited to, sodium dodecyl sulfate (SDS), digitonin, leucoperm, saponin, and tween20. In some embodiments, suitable detergents for permeabilizing nuclei include, but are not limited to, non-ionic detergents, Triton X-100, Nonidet-P40, ionic detergents, sodium dodecyl sulfate (SDS), deoxycholate, sarkosyl, and additional detergents identifiable by those of skill in the art. Suitable concentrations of detergents for permeabilizing cells and organelles such as nuclei include various concentrations depending on the detergent (see, for example, sodium dodecyl sulfate at a final concentration of up to 1%).Additional information on common detergents, including their critical micelle concentration values ​​(CMC) and other properties, can be found in “Detergents: Handbook & Selection Guide to Detergents & Detergent Removal” available from G-Biosciences (2018), Neugebauer, Detergents: An overview, in Methods in Enzymology, MP Deutscher, Editor (1990) Academic Press. p. 239-253, and Schramm et al., Surfactants and their applications. Annual Reports Section “C” (Physical Chemistry), 2003.99(0): p. 3-48. Such information allows the skilled user to easily fine-tune the detergent concentration to ensure that the CMC is not exceeded and that complete lysis of the cells / organelles is not caused. As will be appreciated by those of skill in the art, in the embodiments described herein, permeabilization allows enzymes and other reagents to passively pass through the cell membrane and carry out enzymatic reactions within the cell or organelle, while cellular material, such as nucleic acids (e.g., mRNA and genomic DNA), remains trapped within the cell or nucleus and does not diffuse out.

[0042] In an embodiment of the method described herein, after permeabilization, the cell or organelle, e.g., nucleus, is contacted with a reagent capable of producing a first identifier-tagged nucleic acid inside the cell or organelle, e.g., nucleus, inside the cell source. As summarized above, the first identifier-tagged nucleic acid is generated in the cell source using a template-switching mediated reaction. By "template-switching mediated" reaction is meant a nucleic acid synthesis reaction in which the polymerase switches from the template nucleic acid to the template switch oligonucleotide. Thus, in the method of the present invention, the first identifier-tagged nucleic acid is produced in the cell source using a template switch oligonucleotide and a suitable polymerase. The template switch oligonucleotide is an oligonucleotide that is utilized in a template-switching reaction, e.g., reverse transcription of an RNA template or reverse transcription of a DNA template. Thus, the production of identifier-tagged nucleic acids is driven by template switching and the "template switching" ability of certain nucleic acid polymerases, i.e., using a first nucleic acid strand as a template for polymerization and then switching to a second template nucleic acid strand (sometimes referred to as a "template switch nucleic acid" or "acceptor template") while the polymerization reaction continues. The result is the synthesis of a hybrid nucleic acid strand having a 5' region complementary to the first template nucleic acid strand and a 3' region complementary to the template switch nucleic acid. Methods and reagents related to template switching are also described in U.S. Patent Nos. 9,410,173 and 10,941,397, the disclosures of which are incorporated herein by reference in their entireties.

[0043] The disclosed methods utilize a template switch oligonucleotide in producing a first identifier-tagged nucleic acid by template switching. Thus, an embodiment of the method includes contacting a permeabilized cell source with reagents sufficient to generate a first identifier-tagged nucleic acid via a template-switching reaction in the cell source, such reagents may include a template switch oligonucleotide, a template-switching polymerase, a first strand primer, etc., as described in more detail below.

[0044] "Template switch oligonucleotide" refers to an oligonucleotide template that a polymerase switches from an initial template (e.g., a template nucleic acid (e.g., an RNA template or a DNA template)) during a nucleic acid polymerization reaction. In this regard, the template may be referred to as a "donor template" and the template switch oligonucleotide may be referred to as an "acceptor template." A template switch oligonucleotide may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring. For example, a template switch oligonucleotide may include one or more nucleotide analogs (e.g., LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, etc.), linkage modifications (e.g., phosphorothioate, 3'-3' and 5'-5' back linkages), 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 a desired function to the template switch oligonucleotide.

[0045] In certain aspects, the template switch oligonucleotide comprises a 3' hybridization domain located at its 3' end. The 3' hybridization domain can vary in length, in some cases ranging from 2-10 nucleotides in length, e.g., 3-7 nt in length. The 3' hybridization domain of the template switch oligonucleotide can comprise a sequence complementary to a non-templated sequence, e.g., a deoxycytidine stretch added to the 3' end of the newly synthesized reverse transcribed first strand cDNA. A non-templated sequence, described in more detail below, generally refers to a sequence that does not correspond to a template, e.g., an RNA template or a DNA template, and is not templated. When present in the 3' hybridization domain of a template switch oligonucleotide, the non-templated sequence can encompass the entire 3' hybridization domain or a portion thereof. In some cases, the non-templated sequence may comprise or consist of a hetero-polynucleotide, and the length of such a hetero-polynucleotide may vary between 2-10 nt, for example, 3-7 nt in length, including 3 nt. In some cases, the non-templated sequence may comprise or consist of a homo-polynucleotide, and the length of such a homo-polynucleotide may vary between 2-10 nt, for example, 3-7 nt in length, including 3 nt. According to some embodiments, the polymerase (e.g., a reverse transcriptase such as MMLV RT) combined in the reaction mixture has terminal transferase activity such that a homo-nucleotide extension (e.g., a homo-trinucleotide such as CCC) can be added to the 3' end of the nascent strand, and the 3' hybridization domain of the template switch oligonucleotide comprises a homo-nucleotide extension complementary to the homo-nucleotide extension (e.g., a homo-trinucleotide such as GGG) at the 3' end of the nascent strand.In other aspects, where a polymerase with terminal transferase activity adds a nucleotide extension (e.g., a trinucleotide extension) to the 3' end of the nascent strand, the 3' hybridization domain of the template switch oligonucleotide comprises a hetero-trinucleotide extension comprising cytosine and guanine (e.g., a r(C / G)3 oligonucleotide), and the hetero-trinucleotide extension 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.

[0046] In addition to the 3' hybridization domain (located at the 3' end of the template switch oligonucleotide), the template switch oligonucleotide further comprises a first identifier domain, e.g., as described above. In some embodiments, the first identifier domain is located on the template switch oligonucleotide at a position 3' of the 5' end, and thus 5' of the 3' hybridization domain. For all of the given cell sources of the first set of subportions, the same template switch oligonucleotide with the same first identifier domain is used in the template switching-mediated reaction. Thus, the first identifier-tagged nucleic acids produced in each cell source of each subportion have the same or a common first identifier domain. However, because the template switch oligonucleotides used with different subportions of the first set differ from each other with respect to at least the sequence of their first identifier domains, the first identifier domain of the tagged nucleic acid of the first subportion can be distinguished from the first identifier domain of the tagged nucleic acid of any other subportion of the first set. Thus, the first identifiers of the template switch oligonucleotides used in different subportions of the first set are identical within a given subportion, but different between the different subportions. Within a given workflow, the number of different template switch oligonucleotides (that differ from each other in terms of the sequence of their first identifiers) can vary and correspond to the number of first subportions of a given first set, and in some cases can range from 2 to 25,000 different template switch oligonucleotides, such as 2 to 25,000 different template switch oligonucleotides, including 96 to 10,000 different template switch oligonucleotides. In general, there should be as many different template switch oligonucleotides as there are first subportions.

[0047] According to some embodiments, the template switch oligonucleotide comprises a modification that prevents a 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, isonucleotide bases (e.g., isocytosine, isoguanine, etc.), and any combination thereof.

[0048] In some cases, the template switch oligonucleotide may include a unique molecular identifier. The terms "unique molecular identifier" and "UMI" refer to randomers of various lengths, e.g., ranging from 6-12 nt in length in some cases, that can be used to count individual molecules of a given molecular species. In some cases, counting is facilitated by attaching UMIs to individual molecules of the target of interest from a diverse pool of UMIs, such that each individual molecule receives a unique UMI. In such cases, by counting individual transcript molecules, PCR biases generated during NGS library preparation can be corrected and a more quantitative understanding of the sample population can be achieved. UMIs may in some cases be used in conjunction with other barcode sequences, such as source barcode sequences (e.g., cell barcode sequences, sample barcode sequences, well barcode sequences, etc.). When present on a template switch oligonucleotide, within a given subportion, a population of different template switch oligonucleotides may be used, which may have the same or a common first identifier domain, but differ from each other with respect to the sequence of their UMI domains. In such cases, the number of different template switch oligonucleotides that differ from one another with respect to their UMI domains but share a common first identifier domain provided in a given subportion can vary.

[0049] In some cases, the template switch oligonucleotide can include an adaptor domain (e.g., a defined nucleotide sequence 5' to the 3' hybridization domain and a first identifier domain of the template switch oligonucleotide). The adaptor domain can serve various purposes in downstream applications. In some cases, the adaptor domain can function as a primer binding site for further amplification or, for example, nested amplification or suppression amplification, for example, to introduce additional domains such as may be used in NGS applications (as described in more detail below). In some cases, the template switch oligonucleotide includes a sequencing platform adaptor construct. By "sequencing platform adaptor construct" is meant a nucleic acid construct that includes at least a portion of a nucleic acid domain (e.g., a sequencing platform adaptor nucleic acid sequence) utilized by a sequencing platform of interest, such as Illumina® (e.g., HiSeq™, MiSeq™, and / or Genome Analyzer™ sequencing systems), Ion Torrent® (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g., the PACBIO RS II sequencing system), Thermo Fisher Scientific (e.g., the SOLiD sequencing system), or any other sequencing platform of interest.In certain aspects, the sequencing platform adaptor construct includes a domain (e.g., a "capture site" or "capture sequence") that specifically binds to a surface-bound sequencing platform oligonucleotide (e.g., a P5 or P7 oligonucleotide bound to the surface of a flow cell in an Illumina® sequencing system); a sequencing primer binding domain (e.g., a domain to which the Read 1 or Read 2 primer of the Illumina® platform may bind); a barcode domain (e.g., a domain that uniquely identifies the sample source of the nucleic acid to be sequenced to enable sample multiplexing by marking all molecules from a given sample with a specific barcode or "tag"); a barcode sequencing primer binding domain (a domain to which the primers used for barcode sequencing bind); a molecular identification domain (e.g., a molecular index tag such as a randomized tag of 4, 6, or other number of nucleotides) to uniquely mark molecules of interest to determine expression levels based on the number of instances in which the unique tag is sequenced; or any combination of such domains. In certain aspects, the barcode domain (e.g., sample index tag) and the molecular identification domain (e.g., molecular index tag) may be included in the same nucleic acid. A sequencing platform adaptor construct can include a nucleic acid domain (e.g., a "sequencing adaptor") of any length and sequence suitable for a sequencing platform of interest. In certain aspects, the nucleic acid domain is between 4 and 200 nucleotides in length. For example, the nucleic acid domain can be between 4 and 100 nucleotides in length, e.g., between 6 and 75, 8 and 50, or between 10 and 40 nucleotides in length. According to certain embodiments, a sequencing platform adaptor construct includes a nucleic acid domain that is between 2 and 8 nucleotides in length, e.g., between 9 and 15 nucleotides in length, between 16 and 22 nucleotides in length, between 23 and 29 nucleotides in length, or between 30 and 36 nucleotides in length.Examples of such adaptor domains, including possible sequencing platform adaptor constructs, include, but are not limited to, those described in U.S. Pat. Nos. 9,719,136, 10,415,087, 10,781,443, 10,941,397, 10,954,510, and 11,124,828, the disclosures of which are incorporated herein by reference.

[0050] In certain aspects, the sequencing platform adaptor construct comprises a nucleic acid domain that is a domain (e.g., a "capture site" or "capture sequence") that specifically binds to a surface-bound sequencing platform oligonucleotide (e.g., a P5 or P7 oligonucleotide bound to the surface of a flow cell in an Illumina® sequencing system), a sequencing primer binding domain (e.g., a domain to which a read 1 or read 2 primer of an Illumina® platform may bind). The sequencing platform adaptor construct may comprise a nucleic acid domain (e.g., a "sequencing adaptor") of any length and sequence suitable for the sequencing platform of interest. In certain aspects, the nucleic acid domain is 4-200 nt in length. For example, the nucleic acid domain may be 4-100 nt in length, e.g., 6-75, 8-50, or 10-40 nt in length. 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 nucleotides in length, 16-22 nt in length, 23-29 nt in length, or 30-36 nt in length.

[0051] The nucleic acid domain may have a length and sequence that allows a polynucleotide (e.g., an oligonucleotide) used by a sequencing platform of interest to specifically bind to the nucleic acid domain, for example, for solid-phase amplification by synthesis and / or sequencing of cDNA inserts adjacent to the nucleic acid domain. Exemplary nucleic acid domains include the P5 (5'-AATGATACGGCGACCACCGA-3') (SEQ ID NO: 01), P7 (5'-CAAGCAGAAGACGGCATACGAGAT-3') (SEQ ID NO: 02), Lead 1 primer (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') (SEQ ID NO: 03), and Lead 2 primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3') (SEQ ID NO: 04) domains. Other exemplary nucleic acid domains include those in which the A adaptor (5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3') (SEQ ID NO: 05) and the P1 adaptor (5'-CCTCTCTATGGGCAGTCGGTGAT-3') (SEQ ID NO: 06) are used on Ion Torrent™-based sequencing platforms.

[0052] The nucleotide sequence of an adaptor construct useful for sequencing on a sequencing platform of interest may vary and / or change over time. The adaptor sequence is typically provided by the manufacturer of the sequencing platform (e.g., in 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 may be designed to include all or a portion of one or more nucleic acid domains in a configuration that allows sequencing of the nucleic acid insert (corresponding to the template nucleic acid) on the platform of interest. Sequencing platform adaptor constructs that may be included in non-templated sequences, as well as other nucleic acid reagents described herein, are further described in U.S. Patent Application Serial No. 14 / 478,978 (published as US2015-0111789A1 and issued as U.S. Patent No. 10,941,397, the disclosure of which is incorporated herein by reference).

[0053] In addition to the template switch oligonucleotide, for example, the reagent provided to contact the permeabilized cell source of the first set of subportions can include a first strand primer (i.e., a single product nucleic acid primer, for example, for priming synthesis from a template nucleic acid, e.g., an RNA template or a DNA template). The first strand primer (single product nucleic acid primer) includes a template binding domain. For example, the nucleic acid can include a first (e.g., 3') domain configured to hybridize to a template nucleic acid, e.g., an mRNA, ssDNA, etc., and may or may not include a second (e.g., 5') domain that does not hybridize to the template nucleic acid, e.g., one or more additional domains that may be considered as non-template sequence domains, e.g., as described in more detail below. The sequence of the template binding domain can be independently defined or arbitrary. In certain aspects, the template binding domain has a defined sequence, e.g., poly dT or a gene-specific sequence. In other aspects, the template binding domain has an arbitrary sequence (e.g., a random sequence, such as a random hexamer sequence). In still other cases, the template binding domain may be quasi-random, for example, as described in U.S. Patent No. 8,206,913, the disclosure of which is incorporated herein by reference. 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. The first strand primer may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring. For example, the single product nucleic acid primer may include one or more nucleotide analogs (e.g., LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, etc.), linkage modifications (e.g., phosphorothioate, 3'-3' and 5'-5' reverse linkages), 5' and / or 3' end modifications (e.g., 5' and / or 3' amino, biotin, DIG, phosphate, thiol, dye, quencher, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired function to the single product nucleic acid primer.In some cases, the first strand primer (i.e., the single product nucleic acid primer) may include an adapter domain (e.g., a defined nucleotide sequence 5' of the 3' template binding domain of the single product nucleic acid primer), which may serve various purposes in downstream applications. In some cases, the adapter domain may function as a primer binding site for further amplification, as described herein.

[0054] In addition to the template switch oligonucleotide and the first strand primer, the reagents provided to contact, for example, the permeabilized cell source of the first set of subportions may include a polymerase capable of template switching, which uses the first nucleic acid strand as a template for polymerization and then switches to the 3' end of the second template nucleic acid strand, i.e., continues the same polymerization reaction. In some cases, the polymerase capable of template switching is a reverse transcriptase. Reverse transcriptases capable of template switching that can be used to practice the methods of the present invention 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, variant derivatives, or functional fragments thereof, such as RNase H minus or RNase H reductase. For example, the reverse transcriptase may be Moloney murine leukemia virus reverse transcriptase (MMLV RT) or Bombyx mori reverse transcriptase (e.g., Bombyx mori R2 non-LTR element reverse transcriptase). Template-switching capable polymerases useful for carrying out the subject methods are commercially available and include SMARTScribe™ reverse transcriptase and PrimeScript™ reverse transcriptase available from Takara Bio USA (San Jose, Calif.). In addition to template-switching capabilities, polymerases may contain other useful functions. For example, polymerases may have terminal transferase activity, where the polymerase can catalyze the addition of deoxyribonucleotides to the 3' hydroxyl end of an RNA or DNA molecule. In certain embodiments, when the polymerase reaches the 5' end of the template, the polymerase can incorporate one or more additional nucleotides at the 3' end of the nascent strand that are not encoded by the template. For example, if the polymerase has terminal transferase activity, the polymerase may be able to incorporate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional nucleotides at the 3' end of the nascent strand.All of the nucleotides can be the same (e.g., creating a homonucleotide extension at the 3' end of the nascent strand), or one or more of the nucleotides can be different from the other nucleotides (e.g., creating a heteronucleotide extension at the 3' end of the nascent strand). In certain aspects, the terminal transferase activity of the polymerase adds a homonucleotide extension of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the same nucleotides (e.g., all dCTPs, all dGTPs, all dATPs, or all dTTPs). For example, according to one embodiment, the polymerase is MMLV reverse transcriptase (MMLV RT). MMLV RT incorporates additional nucleotides (mainly dCTPs, e.g., three dCTPs) at the 3' end of the nascent strand. As described in more detail elsewhere herein, these additional nucleotides can be useful, for example, to allow hybridization between the 3' hybridization domain of the template switch oligonucleotide and the 3' end of the nascent strand to facilitate template switching by a polymerase from the template to the template switch oligonucleotide.

[0055] The template nucleic acid of the cellular source from which the first identifier-tagged nucleic acid is produced in the cellular source may vary. According to certain embodiments, the template nucleic acid is a template ribonucleic acid (template RNA). Template RNA can be messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), transactional small interfering RNA (ta-siRNA), natural interfering RNA (nat-siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long noncoding RNA (lncRNA), noncoding RNA (ncRNA), transfer-messenger RNA (tmRNA), precursor messenger RNA (pre-mRNA), small Cajal body-specific RNA (scaRNA), piwi-interacting RNA (piRNA), endoribonuclease-prepared siRNA (esiRNA), short transient (small The template nucleic acid may be any type of RNA (or subtype thereof), including but not limited to, stRNA, signal recognition RNA, telomeric RNA, ribozyme, or any combination of those RNA types or subtypes. According to certain embodiments, the template nucleic acid is a template deoxyribonucleic acid (template DNA). The template DNA may be any type of DNA (or subtype thereof), including but not limited to, genomic DNA (e.g., prokaryotic genomic DNA (e.g., bacterial genomic DNA, archaeal genomic DNA, etc.), eukaryotic genomic DNA (e.g., plant genomic DNA, fungal genomic DNA, animal genomic DNA (e.g., mammalian genomic DNA (e.g., human genomic DNA, rodent genomic DNA (e.g., mouse, rat)), insect genomic DNA (e.g., Drosophila), amphibian genomic DNA (e.g., Xenopus), etc.)), viral genomic DNA, mitochondrial DNA, or any combination of those DNA types or subtypes.

[0056] The template switching reaction as described above produces a first set of first identifier-tagged cellular source subportions, each cellular source subportion comprising a plurality of cellular sources, the plurality of cellular sources housing or containing (i.e., including) a first identifier-tagged nucleic acid, e.g., a reverse transcription product nucleic acid comprising a first identifier domain. As outlined above, the first identifier of the first identifier-tagged nucleic acid in the cellular source of a given subportion is the same or common, since it was provided by the template switch oligo having that first identifier. However, within any two subportions of the first set, the first identifier of the tagged nucleic acid is different, so that the tagged nucleic acid of the first subportion can be distinguished from the tagged nucleic acid of any other subportion of the first set.

[0057] The reagents used in the generation of the first identifier-tagged nucleic acids in the first set of sub-portion cell sources can be provided to the cell sources using any convenient protocol. For example, as indicated above, the reagents can be present in the sub-portion containers (e.g., wells) in dry or liquid form, as needed, prior to introducing the cell sources into the containers. Alternatively, the reagents can be provided to the sub-portion containers housing the cell sources, for example, by manually introducing them into the containers, by dispensing them into the containers using, for example, an automated liquid dispensing system, or the like. In some embodiments, a multi-sample nanodispenser (MSND) system is used, including a multi-well plate, for example, in the form of an array of addressable nanowells, and a sample dispenser. An example of such an MSND system is the ICELL8® Single-Cell MSND System (Takara Bio USA, San Jose, CA). Further details of the ICELL8® MSND system can be found in U.S. Pat. Nos. 7,833,709 and 8,252,581, and published U.S. Patent Application Publication Nos. 2015 / 0362420 and 2016 / 0245813, the disclosures of which are incorporated herein by reference.

[0058] Pooling / Redistribution After production of the first set of first identifier-tagged cell source subportions, the first identifier-tagged cell source subportions are combined or pooled to generate a first cell source pool that includes the first identifier-tagged nucleic acid. The different subportion cell sources may be combined or pooled using any convenient protocol. The number of cell sources in the resulting first cell source pool may vary, and in some cases ranges from 2 to 10,000,000 cells, such as 10,000 to 1,000,000 cells or 10,000 to 100,000 cells.

[0059] After pooling, the resulting first cell source pool is allocated into a second set of subportions, each of which comprises a plurality of cell sources comprising the first identifier-tagged nucleic acid. In other words, the first cell source pool is divided or separated into a plurality of subportions, which collectively constitute the second set of subportions, with different subportions comprising a plurality of or multiple cell sources, and the multiple cell sources comprising the different subportions comprising the first identifier-tagged nucleic acid, for example, as described above. The number of subportions comprising the second set can vary, but in some cases, the number ranges from 2 to 25,000 subportions, such as 96 to 10,000 subportions (including 96 to 5,184 subportions). In some cases, the number of subportions in the second set is the same as the number of subportions in the first set. As indicated above, each subportion of the second set is composed of a plurality of cell sources, i.e., comprises a plurality of cell sources. The number of cell sources that make up a given subportion of the second set can vary, but in some cases the number ranges from 1 to 10,000, such as from 100 to 1,000, including from 100 to 500.

[0060] Within a given sub-portion of the second set, the multiple cell sources constituting the sub-portion are each different in terms of the first identifier domain of the first identifier-tagged nucleic acid present in the cell source. For the pooling / redistribution step, the cell sources from different sub-portions of the first set are combined into the same sub-portion of the second set. Within this same sub-portion, the first identifier-tagged nucleic acids in the cell sources are different from each other in terms of the sequence of the first identifier domain. Thus, a given sub-portion of the second set has multiple different first identifier domains, each different domain present in its own cell source.

[0061] Production of nucleic acids that can be identified from cell origin In one embodiment of the present invention, after allocating the first set of pooled cell sources to a second set of sub-portions, cell-source-identifiable nucleic acids are then produced from the multiple cell sources in the sub-portions of the second set. As described above, a cell-source-identifiable nucleic acid is a nucleic acid whose origin or source can be determined based on an identifier sequence present in the nucleic acid, the identifier sequence comprising at least a first and a second identifier sequence. Thus, the cell-source-identifiable nucleic acid comprises both a first identifier and a second identifier, and the sequence information obtained from the combination of these allows for determining the source or source from which the cell-source-identifiable nucleic acid was prepared, i.e. the starting cell source. As will be discussed in more detail below, the second identifier may be comprised of a single domain of consecutive nucleotides, or may be comprised of two or more, e.g., a first and a second, different sub-identifier domains, depending on, for example, the protocol used to prepare the cell-source-identifiable nucleic acid. In each sub-portion, the second identifier present on the cell-source-identifiable nucleic acid is identical. Furthermore, the second identifiers of the different sub-portions of the second set are different. Thus, the second identifier of each subportion of the second set is identical within a given subportion, but different between different subportions. Thus, the cell source-identifiable nucleic acids of different subportions of the second set can be distinguished from each other by their second identifiers. In some embodiments, the combination of the second identifier associated with the nucleic acid in the second set of subportions and the first identifier associated with the nucleic acid in the first set of subportions confers a unique combination of the first and second identifiers to each nucleic acid collection generated from a given cell source that identifies the cell source of those nucleic acids. In other embodiments, the combination of the first identifier, the second identifier, and any third or additional identifiers added in the additional rounds of indexing confers a unique identification to the nucleic acid derived from a particular cell source from the initial multiple cell sources.

[0062] By selecting an appropriate number of initial cell sources and different first and second identifiers (and corresponding first and second sub-portions), or optionally third or further identifiers from subsequent rounds of indexing, one can readily provide a number of combinations in which the probability of nucleic acids derived from two different cell sources having identical first and second identifiers is negligible, i.e., close to zero, or less than 5%, or less than 2%, less than 1%, less than 0.1%, or less than 0.01%.

[0063] As described above, the second identifier incorporated into the first identifier-tagged nucleic acid in the second set of subportions may be composed of a single domain of consecutive nucleotides or may be composed of two or more, e.g., a first and a second, different sub-identifier domains, depending on, e.g., the protocol used to prepare the cell source-identifiable nucleic acid. Thus, in some cases, the second identifier may be composed of a single domain of consecutive nucleotides, and the length of such a domain may vary in some cases, ranging from 4 to 20, e.g., 8 to 12. The second identifier of this embodiment may be introduced in several different ways, e.g., by a primer in an amplification reaction (which may include one or more amplification rounds, e.g., one or more PCR rounds), as part of a ligation adaptor, via tagmentation, etc. In still other cases, the second identifier is composed of two or more, e.g., a first and a second, different sub-identifier domains. The length of these sub-identifier domains may vary in some cases, ranging from 4 to 20, e.g., 8 to 12. The second identifier in this embodiment can be introduced in several different ways, for example, by a primer in an amplification reaction (which may include one or more amplification rounds, e.g., one or more PCR rounds), as part of a ligation adaptor, via tagmentation, etc. In those embodiments in which two or more, e.g., a first and a second sub-identifier, are used to constitute the second identifier, the same combination of sub-identifiers is used for a given sub-population, and a different combination of sub-identifiers is used for a different sub-population. However, a given first sub-identifier need not be used for only one sub-population. Instead, the same first sub-identifier can be used in different sub-populations, as long as it is paired with a different second sub-identifier within each sub-population, such that the combination of the first and second sub-identifiers of a given sub-population distinguishes the other sub-populations of the second set.In this manner, the total collection of sub-identifiers used to produce cell source-identifiable nucleic acids may be less than the total number of subpopulations in the second set, and in some cases the total number of sub-identifiers is 1-30%, e.g., 1-25%, or 3-10%, of the total number of subpopulations in the second set.

[0064] If desired, prior to the production of cell source-identifiable nucleic acids, nucleic acids can be released from the cell sources in the second set of subpopulations, for example, by lysing the cell sources. Lysis can be achieved, for example, by heating or freeze-thawing the cell sources, or by using detergents or other chemical methods, or a combination thereof. However, any suitable lysis method can be used. In some cases, a mild lysis procedure can be advantageously used to prevent the release of nuclear chromatin, thereby avoiding genomic contamination of the cDNA library and minimizing degradation of mRNA. For example, heating cells at 72°C for 2 minutes in the presence of Tween-20 is sufficient to lyse the cells while not resulting in 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 to 70° C. for 90 seconds in PCR buffer II (Applied Biosystems) supplemented with 0.5% NP-40 (Kurimoto et al., Nucleic Acids Res 34(5):e42 (2006)), or lysis can be achieved with a protease such as proteinase K, or by the use of a chaotropic salt such as guanidine isothiocyanate (U.S. Publication No. 2007 / 0281313).

[0065] As described above, the cell source-identifiable nucleic acid may be produced in the second set of subportions using any convenient protocol that associates the second identifier (including when the second identifier comprises the first and second sub-identifiers) with the first identifier-tagged nucleic acid of the subportion to produce a cell source-identifiable nucleic acid that comprises both the first and second identifiers. Examples of such protocols include, but are not limited to, amplification protocols, ligation protocols, tagmentation protocols, second strand synthesis reactions, and the like. The reaction mixture components in such protocols are combined under conditions sufficient to produce the desired cell source-identifiable nucleic acid product of the reaction. For example, in some cases, the reaction components of an amplification reaction are combined under conditions sufficient to produce a cell source-identifiable nucleic acid product via one or more amplification rounds, e.g., one or more PCR rounds. In some cases, the reaction components of a ligation reaction are combined under conditions sufficient to produce a ligated cell source-identifiable product nucleic acid. In yet other cases, reaction components of a tagmentation reaction are combined under conditions sufficient to produce a cell-source-identifiable tagmentation nucleic acid that may or may not be used or subjected to a subsequent reaction step.

[0066] The prepared reaction mixture provides the components necessary to generate conditions sufficient to produce the desired cell-source-identifiable product nucleic acid. "Conditions sufficient to produce the desired cell-source-identifiable nucleic acid" refers to reaction conditions that allow the relevant nucleic acids in the reaction and / or other reaction components 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 chemical processes, such as polymerization, hydrolysis, ligation, tagmentation, etc. Achieving suitable reaction conditions may include selecting reaction mixture components, their concentrations, and reaction temperatures to create an environment in which relevant processes proceed, including, for example, relevant nucleic acids hybridizing to each other in a sequence-specific manner, relevant polymerases resulting in the extension of nucleic acids, etc. In addition to the particular nucleic acids of the reaction (e.g., template nucleic acid, oligonucleotides, primers, etc.), the reaction mixture may include buffer components that establish the appropriate pH, salt concentration (e.g., KCl concentration), etc. Conditions sufficient for the formation of double-stranded nucleic acid complexes can include conditions suitable for hybridization (also referred to as "hybridization conditions").

[0067] Achieving suitable reaction conditions can include selecting reaction mixture components, their concentrations, and reaction temperature to create an environment in which one or more polymerases are active and / or in which the associated nucleic acids in the reaction interact (e.g., hybridize) with one another in a desired manner. In suitable reaction conditions, in addition to the reaction components, the reaction mixture contains the appropriate pH, salt concentration (e.g., KCl concentration), metal cofactor concentration (e.g., MgCl concentration), and / or pH for the extension reaction, e.g., second strand synthesis reaction, and / or template switching to occur. 2+ or Mn 2+Other components may include one or more nuclease inhibitors (e.g., RNase inhibitors and / or DNase inhibitors), one or more additives to facilitate amplification / replication of GC-rich sequences (e.g., GC-Melt™ Reagent (Takara Bio USA, Inc., San Jose, 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 facilitating polymerase-mediated extension reactions and / or template switching, etc.

[0068] One or more reaction mixtures may have a pH suitable for amplification (e.g., PCR amplification), ligation, second strand synthesis, or tagmentation. In certain embodiments, the pH of the reaction mixture is in the range of 5 to 9, e.g., 7 to 9, 8 to 9, e.g., 8 to 8.5. In some cases, the reaction mixture includes a pH adjuster. pH adjusters of interest include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphate buffer, citrate buffer, and the like. For example, the pH of the reaction mixture can be adjusted to a desired range by adding an appropriate amount of a pH adjuster.

[0069] Suitable temperature ranges for primer extension reactions can vary according to factors such as the particular polymerase used, the melting temperature (Tm) 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 ranging from 4°C to 72°C, e.g., 16°C to 70°C, e.g., 37°C to 50°C, e.g., 40°C to 45°C, inclusive.

[0070] As summarized above, the second identifier sequence may be associated with the first identifier-tagged nucleic acid by various means, such as through an amplification-mediated reaction, a ligation-mediated reaction, a tagmentation-mediated reaction, a second strand synthesis reaction, an isothermal amplification reaction, a template switching reaction, etc. For example, the second identifier sequence present on a primer or oligonucleotide may be incorporated into the first identifier-tagged nucleic acid during an amplification reaction. In some cases, the second identifier sequence may be directly attached to the first identifier-tagged nucleic acid. Methods for directly attaching a non-templated sequence to a nucleic acid are varied and may include, but are not limited to, ligation, chemical synthesis / ligation, enzymatic nucleotide addition (e.g., by a polymerase with terminal transferase activity), etc. In still other cases, the second identifier sequence may be associated with the first identifier-tagged nucleic acid using tagmentation.

[0071] When amplification is used, the association of the second identifier with the first identifier-tagged nucleic acid in the subportion of the second set may utilize one or more amplification rounds, e.g., PCR rounds, and primers, e.g., forward and reverse amplification primers, may be used in combination with a suitable amplification polymerase to produce a cell source-identifiable nucleic acid comprising a first and a second identifier domain, and the second identifier domain may be comprised of a single contiguous domain or two or more subdomains. In such an embodiment, the primer may comprise a primer binding site configured to hybridize to a complementary site of the first identifier-tagged nucleic acid. The primer used in the amplification embodiment has a template-binding domain that hybridizes to a corresponding domain in the first identifier-tagged nucleic acid. This template-binding domain may be defined as, e.g., gene-specific, arbitrary, e.g., random, quasi-random, etc., as described above. A given primer used in one or more amplification rounds may comprise a second identifier component, e.g., an entire domain or a sub-identifier thereof. For example, when the second identifier is composed of a first and a second sub-identifier, each of the first and second primers may include one of the sub-identifiers, for example, the 5' position of the primer binding site of the primer. In addition, the primer used in the amplification-mediated reaction may include one or more additional domains as desired. Such additional domains often include, but are not limited to, an adapter domain (e.g., a sequencing platform adapter construct) as described above.

[0072] The amplification-mediated reaction for producing a cell source-identifiable nucleic acid may also use a polymerase suitable for use in amplifying, for example, a primed first identifier-tagged nucleic acid. 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, Terra™ DNA polymerase, those described in U.S. Pat. No. 6,127,155, the disclosure of which is incorporated herein by reference in its entirety, and derivatives thereof. In some cases, the amplification polymerase may be a hot-start polymerase, including, but not limited to, hot-start Taq DNA polymerase, hot-start Pfu DNA polymerase, and the like. The amplification polymerase may be combined in a reaction mixture such that the final concentration of the amplification polymerase is sufficient to produce a desired amount of product nucleic acid. 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 / μL (U / μL), including, for example, 0.5-100 U / μL, for example, 1-50 U / μL, for example, 5-25 U / μL, for example, 20 U / μL. The nucleic acid reaction, e.g., the amplification reaction, of the subject method can include combining dNTPs to the reaction mixture. In certain embodiments, each of the four naturally occurring dNTPs (dATP, dGTP, dCTP, and dTTP) is added to the reaction mixture. For example, dATP, dGTP, dCTP, and dTTP can be added to the reaction mixture such that the final concentration of each dNTP is 0.01-100 mM, for example, 0.1-10 mM, including 0.5-5 mM (e.g., 1 mM). In some cases, one or more types of nucleotides added to the reaction mixture may be non-naturally occurring nucleotides, e.g., modified nucleotides having linkages or other moieties attached thereto (e.g., fluorescent moieties, biotin moieties), nucleotide analogs, or any other type of non-naturally occurring nucleotide that finds use in a subject method or downstream application of interest.

[0073] The reaction mixture may be subjected to various temperatures to drive various aspects of the reaction, including, but not limited to, for example, denaturation / melting of the nucleic acid, hybridization / annealing of the nucleic acid, polymerase-mediated elongation / extension, etc. The temperatures at which various processes are carried out may be referenced according to the process occurring, including, for example, melting temperature, annealing temperature, elongation temperature, etc. Optimal temperatures for such processes vary, for example, depending on the polymerase used, on the characteristics of the nucleic acid, etc. Optimal temperatures for particular polymerases, including reverse transcriptases and amplification polymerases, may be readily obtained from reference texts. Optimal temperatures associated with nucleic acids, for example, annealing and melting temperatures, may be readily calculated based on known properties of the subject nucleic acid, including, for example, total length, hybridization length, G / C content, secondary structure predictions, etc.

[0074] As described above, the amplification-mediated reaction for associating the second identifier with the first identifier-tagged nucleic acid to produce a cell source-identifiable nucleic acid may include one or more amplification rounds, e.g., PCR application rounds. For example, each first round primer may include a different subdomain of the second identifier, and amplification of the first identifier-tagged nucleic acid with such primers produces an amplicon that includes not only the first identifier, but also the first and second subdomains that collectively constitute the second identifier. Alternatively, the first amplification round may be employed with primers that amplify a desired fraction of the first identifier-tagged nucleic acid, e.g., the amplification is performed with primers that include a gene-specific template binding domain. After this first round, a second round of primers that introduce the first and second subdomains may be performed to produce a cell source-identifiable nucleic acid. The number of surrounding amplifications used in a given workflow may vary as desired.

[0075] In certain aspects, the second identifier sequence is associated with the first identifier-tagged nucleic acid using a ligation protocol. In these cases, the second identifier sequence can be present on a nucleic acid that is ligated to the end of the first identifier-tagged nucleic acid. Any convenient ligase can be used, such as T4 ligase. In some cases, the second identifier can be incorporated into a stem-loop adaptor construct that is ligated to the first identifier-tagged nucleic acid. Further details regarding such adaptors are provided in U.S. Patent Nos. 7,803,550, 8,071,312, 8,399,199, 8,728,737, 9,598,727, 10,196,686, 10,208,337, and 11,072,823, the disclosures of which are incorporated herein by reference.

[0076] In yet another embodiment, the method utilizes a tagmentation reaction, which may include, for example, the use of tagmentation reaction components, to associate a second identifier sequence with a first identifier-tagged nucleic acid. The reaction components and tagmentation process used may vary as needed. The transposome used for tagmentation may include a transposon nucleic acid that includes a transposase and a transposon end domain and a second identifier sequence, for example, a second sub-identifier domain. These domains may be one of the same sequence or different sequences, as required by the researcher to be functionally defined. The domains may also overlap, such that a portion of the second identifier sequence domain may be present in the transposon end domain. Tagmentation processes, transposition-based sequence manipulations, and components that can be used in tagmentation or transposition-based reactions are described, for example, in U.S. Patent Nos. 10,017,759, 9,790,476, 9,683,230, 9,388,465, 9,238,671, 9,193,999, 8,383,345, 6,294,385, 6,159,736, 5,869,296, and 5,677,170, the disclosures of which are incorporated herein by reference in their entirety. Various tagmentation processes, and / or one or more components thereof, can be adapted for use in the methods described herein. In some cases, the resulting tagmented sample can be subjected to PCR amplification conditions, for example, using one or more post-tagmentation PCR primers that hybridize to one or more post-tagmentation primer binding sites added during the tagmentation reaction. The post-tagmentation primers can include a non-template sequence, such as, for example, a sequencing platform adaptor construct domain.The non-templated sequence may include any of the nucleic acid domains described elsewhere herein (e.g., a domain that specifically binds to a surface-bound sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular recognition domain, or any combination thereof). Such embodiments find use, for example, where the nucleic acids of a tagmented sample do not include all adapter domains useful or necessary for sequencing on a sequencing platform of interest, with the remaining adapter domains being provided by the primers used to amplify the nucleic acids of the tagmented sample.

[0077] In any protocol used to produce cell source-identifiable nucleic acids, the reagents used in such protocols can be used to introduce additional features into the cell source-identifiable nucleic acid product, and such additional features can be features used in downstream processing of the cell source-identifiable nucleic acid. For example, if the generation of cell source-identifiable nucleic acids is part of a sequencing library generation protocol, it can include additional features incorporated into the cell source-identifiable nucleic acid, such as a primer binding domain that can be used in subsequent amplification rounds, for example, to add a sequencing platform adaptor construct domain, such as a sequencing adaptor platform construct, as described above.

[0078] Representative Embodiments The following sections provide a representative embodiment of the invention in which cell-source-identifiable nucleic acids are prepared ready for use in a sequencing by synthesis NGS protocol. This embodiment is shown diagrammatically in Figures 1A-1D, which illustrate the workflow for producing cell-source-identifiable nucleic acids from initial multiple cell nuclei, although whole cells can be used instead of cell nuclei.

[0079] It is important to note that the integrity of the cell source material remains intact during this protocol. For example, if cells are used in the protocol, the cells remain intact. If cell nuclei are used in the protocol, the nuclei remain intact.

[0080] As illustrated in Figure 1A, multiple cell nuclei are distributed into wells of a 96-well plate. In some embodiments, each well on the 96-well plate receives a cell nucleus, but this is not a requirement of the protocol. Furthermore, the protocol is not limited to the use of 96-well plates, and one of skill in the art will recognize that any suitable container, tube, or vessel will find equivalent use with the present invention, and further, multi-well plates having, for example, 24 wells or 384 wells, or any other multi-well format plate, may all be used with the present invention.

[0081] As shown in Figure 1A, multiple nuclei are distributed into each well of a 96-well plate. For example, typically about 100 to 1,000 nuclei, for example, multiple nuclei such as 100 nuclei, 200 nuclei, or 300 nuclei, are distributed into each well.

[0082] The nuclei are permeabilized either before or after distribution into the wells, allowing a combinatorial indexing reagent, e.g., a template-switching reagent, to enter the nuclei and access the nucleic acid to be analyzed.

[0083] After distributing the cell nuclei into the wells of a 96-well plate, a well-specific template-switch oligonucleotide containing a unique first identifier is distributed into each well. Each well receives a unique well-specific template-switch oligonucleotide (TSO) that differs from the template-switch oligonucleotide distributed into any other well by the sequence of its unique first identifier. Thus, each well receives a different first identifier provided by the well-specific template-switch oligonucleotide delivered to the well. In addition to the template-switching oligonucleotide, a template-switching reagent containing a reverse transcriptase and an oligo-dT primer is also delivered to the well, and a reverse transcription reaction occurs such that a first strand cDNA is produced by poly-A priming, the first strand containing the first identifier sequence of the template-switch oligonucleotide at its 3' end. As a result, each of the nuclei in each well of the multi-well plate contains a cDNA molecule that corresponds to (i.e., is derived from) the mRNA that was present in those cell nuclei. As illustrated in the bottom panel of FIG. 1A, the resulting cDNA molecules are each tagged with a first identifier. That is, each cDNA is a first identifier-tagged nucleic acid, and the first identifiers of the cDNAs in all nuclei in a single well are the same, however, the first identifiers of the cDNAs in nuclei in different wells are different.

[0084] A continuation of this protocol is shown in Figure 1B. As shown in Figure 1B, top left panel, reverse transcription produces a 96-well plate containing cDNA with a well-specific first identifier. That is, this cDNA is a first identifier-tagged nucleic acid. While the first identifier in each nucleus in a single well is identical, the first identifier differs between different wells of the 96-well plate, as represented by the different hatch patterns. The nuclei in each well are collected and then pooled in a single tube and washed to remove the lysed nuclei as well as excess primers and RT reagents (Figure 1B, top right panel). The resulting pooled nuclei are then redistributed into the wells of another 96-well plate with 100 nuclei in each well. The nuclei in each well are then lysed to release the first identifier-tagged cDNA present in the nuclei. As shown in Figure 1B, bottom panel, each well contains a collection of cDNA released from different lysed nuclei, and the first identifiers of the collections from different wells differ from each other.

[0085] This protocol is further illustrated in FIG. 1C. In FIG. 1C, each well of a 96-well plate contains a pool or mixture of first identifier-tagged nucleic acids from a plurality of different original nuclei, as shown, with a plurality of different first identifiers present in each well. A well-unique combination of first and second sub-identifiers, which collectively constitute a second identifier, is then associated with the first identifier-tagged nucleic acids in each of the wells. The unique combination of the first and second sub-identifiers collectively provides a unique second identifier for each well. As shown in FIG. 1C, a first PCR round is used to amplify a subset of the first identifier-tagged nucleic acids using a first gene-specific primer and a primer complementary to an adapter domain, e.g., a lead primer 2 domain, introduced by the TSO. A second amplification round is then used with primers that introduce different sub-domains that collectively constitute the second identifier. A unique second identifier is provided by providing each well with a unique combination of first and second sub-identifiers, the unique combination being provided from a more limited set of sub-identifier domains that are fewer in number than the number of wells. In the illustrated approach, a different first sub-identifier is provided to each column of wells on the plate, and a different second sub-identifier is provided to each row of wells on the plate, such that each well of the plate has its own unique combination of first and second sub-identifiers. Within each well, the first and second sub-identifiers are associated with the first identifier tagged nucleic acid in the well using an amplification-mediated reaction. In the illustrated amplification-mediated reaction, a first PCR round is performed to amplify the gene of interest, e.g., the TCR or BCR gene, on the 3' end (without an adapter) and with an adapter such as RP2 from TSO, as described above, this PCR round using the same primers for all wells. Following this first PCR round, a semi-nested PCR adds the cluster generating sequence P7 to the 5' end (anneals to the RP2 adaptor sequence) and the lead primer 1 and cluster generating sequence P5 to the 3' end (nested gene specific primer). This PCR also adds distinct i5 and i7 indexes (first and second sub-identifiers) to each well.In this case, a combination pattern is used in which all wells in a given column (shown as circles and diamonds) are assigned the same i7 index, and all wells in a given row (shown as stars and hexagons) are assigned the same i5 index. Different i7 indexes are assigned to wells in different columns, and different i5 indexes are assigned to wells in different rows. In this way, a unique combination of different i5 and i7 indexes is assigned to each well, and these indexes function as first and second identifiers. The combination of i5 and i7 indexes collectively associates a unique second identifier with the first identifier tagged nucleic acid of each well. The bottom right panel of FIG. 1C shows the reaction predicted to occur in the top left well shown in the top left plate.

[0086] FIG. 1D illustrates a cell-source-identifiable nucleic acid produced by the protocol shown in FIG. 1A-1C. As shown in FIG. 1D, the cell-source-identifiable nucleic acid is obtained, for example, from the top left well of the 96-well plate shown in FIG. 1C and includes, from left to right, a P5 domain, an i5 domain specific to the top left well, a lead primer 1 domain, the 5' end of the gene amplified by the gene-specific primer amplified in the first PCR round, for example, the TCR or BCR gene, a first identifier (i.e., barcode) provided by the template switch oligonucleotide, a lead primer 2 domain, an i7 domain specific to the top left well, and a P7 domain. All nucleic acids sharing the same combination of i5 index, TSO first identifier, and i7 index can be determined to have been obtained from the same original nucleus. The cell-source-identifiable nucleic acids depicted in FIG. 1D are ready for next generation sequencing (NGS) and after obtaining the sequences, the cell-source-identifiable nucleic acids of the collection that share the same first identifier and i5, i7 indexes can be assigned a cell source, i.e., an originating nucleus, based on at least the first and second identifiers, e.g., as indicated by the first identifier, i5 and i7 indexes of the nucleic acids of the collection.

[0087] Iteration If desired, a given workflow may further include at least one additional pooling / splitting step to produce nucleic acids incorporating at least one additional identifier. For example, after the production of the first identifier-tagged nucleic acid, but before the lysis of the cell source, the cell source containing the identifier-tagged nucleic acid present therein may be pooled and distributed into a set of sub-portions, for example, as described above. An identifier may be associated with the identifier-tagged nucleic acid present in each sub-portion of the cell source by any suitable method, thereby associating another identifier with the identifier-tagged nucleic acid. Any number of additional pooling / splitting steps may be used to provide the final cell source-identifiable nucleic acid with the desired number of different identifiers. Any lysis in a given workflow of such an embodiment is performed on the cell source in the final set of sub-portions. This final step completes the generation of cell source-identifiable nucleic acids that can be identified by their unique combination of identifiers added in each indexing round.

[0088] Further Processing Following production of cell-source-distinguishable nucleic acids in the different subportions of the second set, the different subportions may be pooled, e.g., to combine the different cell-source-distinguishable nucleic acids from two or more (including each) subportions of the second set into a single composition for further processing. The number of different subportions that are combined or pooled in such embodiments may vary, in some cases, from 2 to 25,000 or more, e.g., from 96 to 10,000 (including 384 to 5,184).

[0089] For example, as described, the prepared cell source-identifiable nucleic acid may be further processed as desired, for example, depending on a particular workflow. For example, the cell source-identifiable nucleic acid may be prepared for sequencing applications, for example, next-generation sequencing applications. In such cases, the cell source-identifiable nucleic acid collection constituting the composition may be sequence-ready in that all domains, for example, adapters as described above, are already incorporated into the nucleic acid. For example, sequencing platform adapter constructs that may be required for use in a given sequencing application may be incorporated into the cell source-identifiable nucleic acid during preparation of the cell source-identifiable nucleic acid, for example, by including such on components used in preparation of the cell source-identifiable nucleic acid, such as template switch oligonucleotides, amplification primers, transposon nucleic acids, etc.

[0090] In still other cases, the cell source distinguishable nucleic acid may be further processed to generate a sequence-ready library, and in such cases, any convenient approach may be used. In such embodiments, one or more of such constructs may be incorporated into the cell source distinguishable nucleic acid after its preparation. If desired, such adapter constructs may be added to the nucleic acid of interest, e.g., the cell source distinguishable nucleic acid, by various means. For example, the adapter sequence may be added through the action of a polymerase with terminal transferase activity. The adapter sequence may be incorporated into the nucleic acid during an amplification reaction. In some cases, the adapter sequence may be directly attached to the nucleic acid, e.g., the cell source distinguishable nucleic acid. Methods for directly attaching the adapter sequence to the nucleic acid are various and may include, but are not limited to, ligation, chemical synthesis / ligation, enzymatic nucleotide addition (e.g., by a polymerase with terminal transferase activity), tagmentation, etc.

[0091] In some cases, the method may include attaching a sequencing platform adaptor construct and / or an adaptor comprising any sequence for any use to the end of the nucleic acid. For example, in some cases, the oligonucleotides and / or primers utilized in the subject methods may not include a sequencing platform adaptor construct, and thus the desired sequencing platform adaptor construct may be attached after the production of the cell source-identifiable nucleic acid of interest. The adaptor construct attached to the end of the nucleic acid of interest or a derivative thereof may include any sequence element useful for downstream sequencing applications, including any of the elements described above with respect to the optional sequencing platform adaptor constructs of the oligonucleotides and / or primers of the methods described herein. For example, the adaptor 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-bound sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof.

[0092] The binding of the sequencing platform adaptor construct can be accomplished using any suitable approach. In certain embodiments, the adaptor construct is bound to the end of the product nucleic acid or its derivative using the same or similar approach as a "seamless" cloning strategy. The seamless strategy eliminates one or more rounds of restriction enzyme analysis and digestion, DNA end repair, dephosphorylation, ligation, enzyme inactivation and cleanup, and the loss of the corresponding nucleic acid material. Seamless ligation strategies of interest include the In-Fusion® cloning system available from Takara Bio USA, Inc. (San Jose, Calif.), 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) as described in Zhang et al. (2012) Nucleic Acids Research 40(8):e55, and GeneArt® seamless cloning technology by Life Technologies (Carlsbad, Calif.).

[0093] Any suitable approach can be used to provide additional nucleic acid sequencing domains to a nucleic acid of interest or a derivative thereof that has less than all of the useful or necessary sequencing domains for the sequencing platform of interest. For example, the nucleic acid of interest or a derivative thereof can be amplified using PCR primers with adapter sequences at their 5' ends (e.g., 5' of the region of the primer that is complementary to the nucleic acid of interest or a derivative thereof), so that the amplicon contains not only the adapter sequence in the original nucleic acid, but also the adapter sequence in the primer, in any desired configuration. Other approaches can be used, including those based on seamless cloning strategies, restriction digestion / ligation, tagmentation, etc. Methods for adding nucleic acid domains to next-generation sequencing libraries are known in the art and are described, for example, but not limited to, in U.S. Pat. No. 11,124,828, which is incorporated herein by reference in its entirety.

[0094] For example, after prescribed library preparation and / or amplification steps, as described above, the prepared library may be considered ready for sequencing. In certain embodiments, the provided methods may further include subjecting the prepared library to an NGS protocol. The protocol may be performed on any suitable NGS sequencing platform. NGS sequencing platforms of interest include, but are not limited to, Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ sequencing systems), Ion Torrent® (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g., PACBIO RS II sequencing system), Oxford Nanopore Technologies (ONT), Life Technologies™ (e.g., SOLiD sequencing system), Roche (e.g., 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest. NGS protocols will vary depending on the particular NGS sequencing system used. For example, detailed protocols for sequencing NGS libraries, which may include further amplification (e.g., solid-phase amplification), sequencing of the amplicons, and analysis of the sequencing data, are available from the manufacturer of the NGS sequencing system used.

[0095] Other variations include, for example, replacing the Illumina®-specific sequencing domains with sequencing domains required by a sequencing system from Ion Torrent™ (e.g., the Ion PGM™ and Ion Proton™ sequencing systems), Pacific Biosciences (e.g., the PACBIO RS II sequencing system), Life Technologies™ (e.g., the SOLiD sequencing system), Roche (e.g., the 454GS FLX+ and GS Junior sequencing systems), or any other sequencing platform of interest.

[0096] cell source As mentioned above, the initial cell source from which cell source-identifiable nucleic acids are produced according to embodiments of the present invention may vary. The cell samples from which the cell source may be obtained may come from a variety of sources, including, but not limited to, cell tissue, biopsy, blood sample, cell culture, and the like. Furthermore, the cell samples may come from specific organs, tissues, embryos, blastocysts, tumors, neoplasms, and the like. Without being limited thereto, the number of cell samples that may be analyzed in any embodiment of the present invention may vary based on the desires of the researcher. When multiple cell samples are used, each independent sample is treated as an initial sub-portion of the initial cell source. Furthermore, cells from any population may be the source of the cell source used in the subject methods, such as a population of prokaryotes or eukaryotes, including bacteria or yeast. In some cases, the cell source utilized in the subject methods 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, and the like. In some cases, the mammalian cell sample may be a mammalian blood sample, including, but not limited to, for example, a rodent (e.g., mouse or rat) blood sample, a non-human primate blood sample, a human blood sample, etc.

[0097] In some cases, the cell source utilized in the subject methods may be a lymphocyte source, e.g., an immune cell source including, but not limited to, T cells (e.g., cytotoxic T cells (e.g., CD8+ T cells), helper T cells (e.g., CD4+ T cells), regulatory T cells ("Tregs"), etc.), natural killer (NK) cells, B cells, etc. The subject's immune cells may also include, e.g., peripheral blood mononuclear cells, macrophages, dendritic cells, monocytes, etc.

[0098] In some cases, the source of cells utilized in the subject methods may be derived from plants, including but not limited to monocotyledonous or dicotyledonous plants, such as research plants (e.g., Arabidopsis), as well as fruits (e.g., apple, apricot, avocado, banana, blackberry, blueberry, cantaloupe, coconut, cranberry, date, fig, melon, grapefruit, grape, guava, honeydew, kiwifruit, lemon, lime, mango, nectarine, olive, orange, papaya, passion fruit, peaches, pears, pineapples, plantains, plums, pomegranates, prunes, raspberries, strawberries, tangerines, watermelons, etc.), grains (e.g., barley, beans, canola, corn, cotton, flaxseed, hay, oats, peanuts, rice, sorghum, soybeans, sugar beets, sugar cane, sunflowers, tobacco, wheat, etc.), vegetables (e.g., artichokes, asparagus, beans, beets, bok choy, broccoli, Brussels sprouts, cabbage, carrots, cauliflower, celery, collard greens, etc.), Agricultural plants such as corn-sweet, cucumber, eggplant, endive, greens, kale greens, lettuce, parsley, parsnips, peas, peppers, pumpkin, radish, rhubarb, rutabaga, spinach, squash, sweet potato, tomatillo, tomato, turnip, water chestnut, etc.

[0099] A source of single cells for use in the methods related thereto described herein may be obtained by any convenient method. For example, in some cases, single cells may be obtained by limiting dilution of a cell sample. In some cases, the method may include a step of obtaining 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 connecting cells in a tissue sample, or releasing adherent cells in culture, or mechanically dissociating cells in a sample.

[0100] In some cases, single cells may be obtained by sorting a cell sample using a cell sorting instrument. As used herein, "cell sorter" refers to any instrument that allows for the sorting of individual cells into a container suitable for downstream processing, such as the process of library preparation as described herein. Useful cell sorters include flow cytometers, such as instruments utilized in fluorescence activated cell sorting (FACS). Flow cytometry is a well-known methodology that uses multi-parameter data to identify and differentiate different particle (e.g., cell) types, i.e., particles that vary from other terms such as label (wavelength, intensity), size, etc. in a fluid medium. In flow cytometric analysis of a sample, an aliquot of the sample is first introduced into the flow path of a flow cytometer. Within the flow path, cells in the sample pass through one or more sensing regions substantially one at a time, and each of the cells is separately and individually exposed to a light source at a single wavelength (or in some cases, two or more different light sources), and measurements of scattering and / or fluorescence parameters are recorded separately for each cell, as needed. 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 needed. The cells sorted using a flow cytometer can be sorted into a common container (i.e., a single tube) or can be sorted separately into individual containers. For example, in some cases, the cells can be sorted into individual wells of a multi-well plate, as described below.

[0101] Purpose For example, as described above, the methods of preparing cell-source nucleic acids according to the present invention may be used to prepare sequence-ready libraries for a variety of different purposes. In certain embodiments, the subject methods may be used to generate expression libraries corresponding to mRNA for downstream sequencing on a sequencing platform of interest (e.g., sequencing platforms provided by Illumina®, Ion Torrent™, Pacific Biosciences, Life Technologies™, Roche, etc.).

[0102] The prepared library can be utilized for various downstream analyses, and in some cases, the library preparation can be specifically reconfigured for the desired type of downstream analysis. For example, in some cases, the prepared library can be subjected to whole transcriptome analysis (WTA), including the analysis of non-mRNA RNA species, such as mRNA and non-coding RNA (e.g., long non-coding RNA (lncRNA), non-polyadenylated RNA, snRNA, and snoRNA). Thus, in some cases, the library preparation can be specifically configured to allow the analysis of non-mRNA RNA in the transcriptome, for example, by utilizing primers that do not rely on hybridization to poly(A) tails (e.g., random primers), or by adding poly(A) tails to RNA species that are not naturally polyadenylated prior to the production of product double-stranded cDNA.

[0103] 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. This may be done before any indexing step of the invention, e.g., using RiboGone™ products (Takara Bio USA Inc., San Jose, CA), or after generation of indexed fragments (e.g., ZapR technology as described in U.S. Pat. No. 10,150,985), e.g., after any indexing step, including at the end of all indexing steps before sequencing. Any convenient method of reducing and / or removing unwanted ribosomal RNA may be used for selective removal, including, for example, affinity purification, degradation of contaminating nucleic acids (e.g., using RiboGone™ products (Takara Bio USA Inc., San Jose, CA), and those methods described in U.S. Pat. Nos. 9,428,794 and 10,150,985, the disclosures of which are incorporated herein by reference in their entireties).

[0104] In certain embodiments, the prepared library may be utilized in differential expression analysis, including, for example, where the relative expression (i.e., up- or down-regulation) of one or more genes is determined. 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 a subject differential expression analysis may vary. Differential expression analysis as used herein is not limited with respect to the number of expressed transcripts analyzed in a subject genome. In some embodiments, differential expression analysis may evaluate a limited number of transcripts, such as a panel of marker genes that are specifically targeted for analysis. Alternatively, the entire transcriptome of a cell may be evaluated for differential expression.

[0105] The transcript categories to which targeted expression analysis may be limited are varied and may include, for example, but are not limited to, immune gene transcripts such as cytokines, chemokines, or cell surface markers of immune cell subsets, kinases, G protein-coupled receptors, druggable genes, etc. Useful categories and subcategories of immune genes generally include groups of genes involved in immune system function and successful 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)), including, for example, 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 suppression in mucosa-associated lymphoid tissues, and the like. These include, but are not limited to, immunomodulatory and immunological functions, including but not limited to, immunomodulatory and immunological functions, immunological ...

[0106] In some embodiments, the methods of the invention include preparing an immune cell receptor repertoire library from an RNA sample. Aspects of the subject methods include amplifying immune cell-specific cDNA from product double-stranded cDNA generated from the RNA sample to produce an immune cell receptor repertoire library. An "immune cell receptor repertoire library" generally refers to a nucleic acid library that includes full-length or partial sequences of immune receptors of one or more types of cells or cell populations. For example, immune cell receptor repertoire libraries can be generated for single cells, or for a single cell sample or cell population derived from a single subject, or a population of cell samples (including, for example, a population of samples from two or more subjects). In some cases, the subject libraries can be generated from individual single cells that can be pooled after addition of discriminating nucleic acid sequences.

[0107] As described above, the members of the immune cell receptor repertoire library can vary in length and can be full length or less than full length. In some cases, the members of the library predominantly contain the 5' end of the immune cell receptor. Immune cell receptors of interest include, but are not limited to, T cell receptors (TCRs) and B cell receptors (BCRs).

[0108] In some cases, the immune cell receptor repertoire library may include a TCR repertoire library. The TCR complex is a disulfide-linked membrane-anchored heterodimeric protein that is typically expressed on the surface of T cells and consists of highly variable alpha (α) and beta (β) chains expressed as part of a complex with a CD3 chain molecule. Many natural TCRs exist in heterodimeric αβ or γδ forms. The complete endogenous TCR complex in heterodimeric αβ form includes eight chains: an alpha chain (referred to herein as TCRα or TCR alpha), a beta chain (referred to herein 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 is generated from genetic recombination (VJ recombination of the alpha chain and VDJ recombination of the beta chain), resulting in crossover regions important for antigen (i.e., peptide / MHC) recognition.

[0109] In some cases, a 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 a subject TCR repertoire library 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).

[0110] Where a subject TCR repertoire library member comprises a partial TCR chain sequence, the partial TCR chain sequence may comprise an entire TCR chain variable region or essentially an entire TCR chain variable region (e.g., a TCR alpha chain variable region, a 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 a TCR alpha and / or beta chain 5' mRNA end. In some cases, the resulting library member comprises a sequence from the TCR alpha or beta chain 5' end to at least a portion of the corresponding chain constant region.

[0111] In certain embodiments, the preparation of the immune cell-specific library may include TCR-specific amplification. Such TCR-specific amplification may utilize TCR-specific primers. "TCR-specific primer" refers to a primer that specifically hybridizes to a region of a TCR chain (e.g., TCR alpha chain, TCR beta chain) nucleic acid sequence 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 hybridize to both the TCR alpha chain and the TCR beta chain.

[0112] 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, the human TCR alpha chain constant region, mouse TCR alpha chain constant region, rhesus monkey, hamster, camel, etc.

[0113] An exemplary human TCR alpha chain constant region has the following amino acid sequence: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (sequence number 07). It is encoded by the following nucleic acid sequence: CCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCC AGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA (SEQ ID NO: 08; T cell receptor alpha chain C region, human; GenBank: AY247834.1, AAO72258.1; UniProtKB: P01848).

[0114] An exemplary mouse TCR alpha chain constant region has the following amino acid sequence: PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 09; UniProtKB: P01849), or PNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 10; GenBank: AAA53226.1) These are encoded by the following nucleic acid sequences, respectively: CCATACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGT (SEQ ID NO: 11), and CCAAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGT (SEQ ID NO: 12; GenBank: U07662.1).

[0115] The TCR-specific primers may be designed to specifically hybridize to a TCR beta chain (e.g., TCR beta 1 chain constant region or TCR beta 2 chain constant region) constant region 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, including, for example, a human TCR beta chain constant region, a mouse TCR beta chain constant region, a rhesus monkey, a hamster, a camel, etc.

[0116] An exemplary human TCR beta chain 1 constant region has the following amino acid sequence: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWT QDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO: 13;UniProtKB:P01850;GenBank:CAA25134.1) It is encoded by the following nucleic acid sequence: (SEQ ID NO: 14; GenBank: EF101778.1, X00437.1).

[0117] An exemplary human TCR beta chain 2 constant region has the following amino acid sequence: DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD RAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 15; UniProtKB:A0A5B9, GenBank:AAA60662.1) It is encoded by the following nucleic acid sequence: (SEQ ID NO: 16; GenBank: L34740.1).

[0118] An exemplary mouse TCR beta chain 1 constant region has the following amino acid sequence: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 17; UniProtKB: P01852) It is encoded by the following nucleic acid sequence: (SEQ ID NO: 18; GenBank: FJ188408.1).

[0119] An exemplary mouse TCR beta chain 2 constant region has the following amino acid sequence: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 19, UniProtKB: P01851) It is encoded by the following nucleic acid sequence: (SEQ ID NO: 20; GenBank: U46841.1).

[0120] 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 includes a membrane-bound immunoglobulin (i.e., antibody) binding portion, including a heavy chain and a light chain, each of which contains a constant (C) and a variable (V) region. The immunoglobulin chain of the BCR is linked to the signaling CD79A / B chain by a disulfide bridge. The immunoglobulin chain of the BCR may be of various isotypes, including IgD, IgM, IgA, IgG, or IgE. Similar to the TCR, the immunoglobulin portion of the BCR undergoes V(D)J recombination to generate vast diversity within the population.

[0121] In some cases, an immune cell receptor repertoire library may include a BCR repertoire library, for example, a BCR repertoire library may include BCR immunoglobulin chain sequences (e.g., including IgD, IgM, IgA, IgG, or IgE chain sequences). The immunoglobulin chain sequences of a subject BCR repertoire library 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).

[0122] When a subject BCR repertoire library member comprises a partial immunoglobulin chain sequence, the partial immunoglobulin chain sequence can comprise an entire immunoglobulin variable region or essentially an entire immunoglobulin variable region (e.g., an immunoglobulin light chain variable region, an immunoglobulin heavy chain variable region). In some cases, the resulting library member comprises an immunoglobulin variable region 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 from the 5' end of an immunoglobulin heavy or light chain to at least a portion of the corresponding immunoglobulin chain constant region.

[0123] In certain embodiments, preparation of immune cell-specific libraries may include BCR-specific amplification (e.g., including immunoglobulin chain-specific amplification). Such immunoglobulin-specific amplification may use immunoglobulin-specific primers. By "immunoglobulin-specific primers" is meant primers that specifically hybridize to a region of an immunoglobulin chain (e.g., immunoglobulin heavy chain, immunoglobulin light chain) nucleic acid sequence or its complement. In some cases, an immunoglobulin-specific primer may hybridize to only one type of immunoglobulin chain, e.g., only immunoglobulin heavy chain, only immunoglobulin light chain, only IgD chain, only IgM chain, only IgA chain, only IgG chain, only IgE chain, etc.

[0124] Immunoglobulin-specific primers can be designed to specifically hybridize to an immunoglobulin heavy chain constant region or its complement. For example, in some cases, an immunoglobulin-specific primer can 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.

[0125] Immunoglobulin-specific primers can be designed to specifically hybridize to an immunoglobulin light chain constant region or its complement. For example, in some cases, immunoglobulin-specific primers can hybridize to a mammalian immunoglobulin light chain constant region or its complement, including, for example, a human immunoglobulin light chain constant region, a mouse immunoglobulin light chain constant region, a rhesus monkey, a hamster, a camel, etc.

[0126] For example, amplifications performed during library preparation, including immune receptor specific amplification, can be performed in a single amplification round, or multiple amplification rounds may be used. For example, in some cases, after the first amplification round, one or more amplification primers not utilized in the first round may be added to the reaction mixture to facilitate a second amplification round using the product of the first amplification round as a nucleic acid template. In some cases, the second or subsequent amplification round may include nested amplification, i.e., where the primer binding site utilized in the second or subsequent amplification round is within (i.e., one or more nucleotides from the 3' or 5' end) of the product generated in the first amplification round. If used, the degree of nesting can vary as desired, including, for example, where the second or subsequent primer binding site is one or more (including two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, etc.) nucleotides from the 3' or 5' end of the amplicon generated in the first amplification round.

[0127] In some cases, the second or subsequent round amplification is not nested, including, for example, when the second amplification round utilizes one or more primer binding sites utilized in a previous amplification round or a primer binding site added during a previous amplification round (e.g., a primer binding site added as part of a non-templated sequence). In some cases, the second or subsequent amplification round may utilize a nested primer amplification site at one end and a non-nested (e.g., a previously used primer binding site or an added primer binding site) primer amplification site at the other end, including when the nested site is at the 3' end of the amplicon or the 5' end of the amplicon.

[0128] Kits, Compositions and Devices Aspects of the present disclosure also include compositions and kits, as well as devices for use therewith or therein.

[0129] Most commonly, the term "kit" is used to describe any assembly of articles that facilitates the performance of a process, method, assay, analysis, sample manipulation, and the like. A kit can contain instructions describing how to use the kit (e.g., instructions describing a method of the invention), chemical reagents or enzymes required for the method, primers, probes, buffers, any type of container (e.g., containers for sample collection or sample manipulation) or reaction vessel, or any other components. A kit need not contain all components required to carry out a method of the invention. Compositions and kits of the invention can include, for example, any one or more of the reaction components described above with respect to the subject methods.

[0130] In some embodiments, the kits of the invention may include a plurality of distinct template switch oligonucleotide compositions each comprising a template switch oligonucleotide comprising a common first identifier, where the first identifiers of the template switch oligonucleotides of the different template switch oligonucleotide compositions are different, and a plurality of distinct second identifier nucleic acids, which may be provided, for example, as subdomains. In such cases, a given template switch oligonucleotide composition may be made from many copies of the same or common template switch oligonucleotide, or a population of different template switch oligonucleotides that share the same or common first identifier sequence, but each differ with respect to the UMI domain. If desired, the different template switch oligonucleotides may be present in different containers, for example, in different wells of a multiplate, including different microwells of a microwell plate. As with the template switch oligonucleotide compositions, the different second identifier nucleic acids may be present in separate containers, for example, different wells of a multiplate, including different microwells of a microwell plate, where the separate containers are different from the container holding the template switch oligonucleotide compositions.

[0131] The kits may further include one or more additional reagents for use in embodiments of the invention, e.g., as described above, including, but not limited to, one or more polymerases (e.g., template-switching polymerases, reverse transcriptases, amplification polymerases, etc.), ligases, transposases, primers, buffers, dNTPs (e.g., including dATP, dCTP, dGTP, dTTP, dUTP, etc., or any one or any combination thereof), etc. The subject kits may include, or compositions and devices may comprise, one or more test reagents, including, e.g., control nucleic acids (e.g., control nucleic acid templates), etc. In some cases, reagents may be provided in lyophilized form, such as lyophilized enzymes (e.g., lyophilized reverse transcriptase, lyophilized DNA polymerase).

[0132] In some cases, the components of the compositions and / or kits of the invention may be presented as a "cocktail", and as used herein, a cocktail refers to a collection or combination of two or more different but similar components in a single container. If desired, the components of the kit may be in separate containers, or multiple components may be in a single container. The subject compositions may be present in any suitable environment. According to one embodiment, the composition is present in a reaction tube (e.g., a 0.2 mL tube, a 0.6 mL tube, a 1.5 mL tube, etc.), or a well or a microfluidic chamber or droplet, or other suitable container. In certain aspects, the composition is present in two or more (e.g., a plurality) reaction tubes or wells (e.g., a plate, e.g., a 96-well plate, a multi-well plate, e.g., containing about 1000, 5000, or 10,000 or more wells). The tubes and / or plates may be made of any suitable material, such as, for example, polypropylene, PDMS, or aluminum. The container may be treated to reduce adsorption of nucleic acids to the walls of the container. In certain aspects, the tubes and / or plates in which the compositions reside provide efficient heat transfer to the compositions (e.g., when placed in a heat block, water bath, thermocycler, etc.) so that the temperature of the composition may be altered within short periods of time, e.g., as needed for a particular enzymatic reaction to occur. According to certain embodiments, the compositions reside in thin-walled polypropylene tubes, or plates with thin-walled polypropylene wells, or materials such as aluminum that have high thermal conductivity.

[0133] In some cases, a collection of individual containers (e.g., separate tubes) or a collection containing multiple containers, e.g., a multi-well device, may contain reagents that may be provided in liquid or dry form.

[0134] Any suitable reaction vessel can be used in the subject kits or devices and / or contain the subject compositions. Useful reaction vessels include, but are not limited to, for example, tubes (e.g., single tubes, multi-tube strips, etc.), wells (e.g., multiwell plates (e.g., 96-well plates, 384-well plates, or plates having any number of wells, such as 2000, 4000, 6000, or 10000 or more). Multiwell plates can be freestanding or part of a chip and / or device, for example, as described in more detail below. Thus, in certain embodiments, the reaction vessel used is one or more wells of a multiwell device. The present disclosure is not limited by the type of multiwell device (e.g., plate or chip) used. In general, such devices have multiple wells that contain or are sized to contain liquid (e.g., liquid confined within the wells such that gravity alone cannot cause the liquid to flow out of the wells). One exemplary chip is the 5184-well SMARTCHIP™ (Takara Bio USA, San Jose, CA). CA). Other exemplary chips are provided in U.S. Patent Nos. 8,252,581, 7,833,709, and 7,547,556, all of which are incorporated herein by reference in their entirety, including, for example, the teachings of chips, wells, thermal cycling conditions, and associated reagents used therein). Other exemplary chips include the OPENARRAY™ plate used in the QUANTSTUDIO™ real-time PCR system (sold by Applied Biosystems). Another exemplary multi-well device is a 96-well or 384-well plate.

[0135] In addition to the above components, the subject kits may further include instructions for using the components of the kit, for example, to practice the subject methods as described above. The instructions are generally recorded on a suitable recording medium. The instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert or on a label of the container of the kit or its components (i.e., associated with the packaging or subpackaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, such as a portable flash drive, CD-ROM, diskette, hard disk drive (HDD), and the like. In still other embodiments, the actual instructions are not present 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 from which the instructions can be downloaded. As with the instructions, this means that the means for obtaining the instructions are recorded on a suitable substrate.

[0136] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0137] Example 1 Analysis of the expression of one or more genes in each of a plurality of cells or nuclei This example is broadly illustrated in Figures 2 and 3. The cells or nuclei are fixed and permeabilized in an appropriate solution (e.g., 1% or 4% paraformaldehyde with NP40 or digitonin). The fixed cells or nuclei are aliquoted into the wells of a multi-well device (e.g., a 96-well or 384-well plate). Alternatively, the wells do not have to be physical wells, but may be composed of droplets, with the cells or nuclei being distributed into the different droplets. The number of cells or nuclei per well or vessel can be varied accordingly depending on the scale of the experiment the researcher wishes to perform. For example, 100 or 1,000 cells per well can be analyzed for each of the 96 or 384 wells of the plate. A reverse transcription mixture including reverse transcriptase, oligo dT, a template switch oligo (TSO) including a first cell-specific barcode (i.e., a well-specific first identifier or first index) and an adapter handle for PCR (i.e., a primer binding site), dNTPs, and buffer salts is added to each of the wells, and the RT reaction is allowed to proceed (e.g., at any suitable temperature, such as 37-50°C, e.g., 42°C, etc., for a time sufficient to complete the reaction, e.g., 60-90 minutes or more). This is shown in step A of FIG. 2. The reaction is stopped, and the cells or nuclei are collected from each of the wells, pooled together, and then redistributed into wells of a second multi-well device. Alternatively, the cells or nuclei may be redistributed into another set of droplets. Optionally, a lysis buffer is added to each of the wells to release the nucleic acid from each of the cells or nuclei. Optionally, the nucleic acid is purified in each well independently. Reagents effective to perform PCR are then added to the wells. These may include a thermostable polymerase, dNTPs, a buffer, and two or more PCR primers, a first primer specific for an adapter handle (primer binding site) sequence present in the TSO used in the reverse transcriptase step, and one or more second primers that have a region at their 3' end that is complementary to either a gene specific sequence (e.g., a TCR constant region gene sequence), a polyA sequence, an adapter handle, or a random sequence, as shown in step B of Figure 2.In one embodiment, each of the two or more PCR primers further contains a barcode sequence (shown as BC2A and BC2B in FIG. 2) that, when combined, provides a second cell-specific barcode sequence (i.e., a second identifier tag). In an alternative embodiment, BC2A or BC2B can be used alone as the second cell-specific barcode sequence (i.e., a second identifier tag). Optionally, the PCR primers may also contain additional sequences for next-generation sequencing. For example, a sequencing platform adaptor construct, such as a lead primer sequence, p5, p7 sequences, etc. Alternatively, these sequencing platform adaptor construct sequences can be added in a second PCR round. This alternative embodiment with a second PCR round is shown in FIG. 3, with the second PCR (PCR2) shown as an additional step C. In this step C, a nested 3' primer is used internally to the secondary primer binding site from step B. This nested primer contains a sequence complementary to the tertiary priming site (5' to the secondary priming site of step B), an optional secondary tag sequence (second subidentifier, BC2B), and an Illumina p7 sequence.

[0138] As shown in Figures 2 and 3, the second identifier tag is added in two parts (BC2A and BC2B). In the embodiment shown in Figure 2, both BC2A and BC2B are added as part of PCR1 (step B of Figure 2). In the embodiment shown in Figure 3, BC2A is added in step B of Figure 3, and BC2B is added similarly in step C.

[0139] The resulting sequencing products are then sequenced using a next-generation sequencing platform to obtain gene sequence information and barcode information (a combination of the first (TSO) barcode (i.e., first identifier) ​​and the second (combined PCR barcode; second identifier)), providing each individual cell or nucleus with a unique cell-specific barcode that can track gene-specific sequences. Thus, the genes or gene expression detected in each of the cells or nuclei of the original collection is determined.

[0140] Example 2: Embodiment of the present invention This example follows the same initial steps as Example 1 up to the redistribution of cells or nuclei into a second multi-well device. This example is generally shown in FIG.

[0141] The cells or nuclei to be analyzed are fixed and permeabilized in an appropriate solution (e.g., 1% or 4% paraformaldehyde with NP40 or digitonin). The fixed cells or nuclei are aliquoted into the wells of a multi-well device (e.g., a 96-well or 384-well plate). Alternatively, instead of physical wells, the cells or nuclei can be distributed into multiple droplets, as known in the art.

[0142] The number of cells or nuclei per well or vessel may vary depending on the scale of the experiment the researcher wishes to perform. For example, 100 or 1,000 cells per well can be used for each of the 96 or 384 wells of a plate. A reverse transcription mixture containing reverse transcriptase, oligo dT, template switch oligo (TSO) containing a first cell-specific barcode (i.e., a well-specific first identifier tag) and an adapter handle for PCR (i.e., a primer binding site), dNTPs, and buffer salts is added to each of the wells, and the RT reaction is allowed to proceed (e.g., at any suitable temperature, such as 37-50°C, e.g., 42°C, for a time sufficient to complete the reaction, e.g., 60-90 minutes or more). The reaction is stopped, and the cells or nuclei are collected from each of the wells, pooled together, and then redistributed into the wells of a second multi-well device. Alternatively, the cells or nuclei may be redistributed into another set of droplets.

[0143] Second strand synthesis and / or isothermal amplification is performed on the redistributed cells or nuclei. At this stage, the redistributed material still remains as intact cells or intact nuclei in the individual wells of the second multi-well device. Second strand synthesis is initiated by adding one or more of the following to the wells: reaction buffer, dNTPs, second strand primers, e.g., primers including a second well-specific barcode (i.e., a second identifier tag) and a second primer binding site, and a polymerase. Second strand synthesis is performed to add a second barcode 5' to the TSO barcode (i.e., the well-specific first identifier tag). Optionally, isothermal amplification is performed by including one or more reverse primers with either a target-specific sequence, an oligo-dT sequence, a sequence complementary to the adapter handle sequence added in the first round, or a random sequence at the 3' end, thereby generating cell-specific nucleic acids with two barcodes. One from each step (template switching step and second strand synthesis step), when combined, can identify which well of the first multiwell device and which well of the second multiwell device in which any particular cell or nucleus was originally located.

[0144] After completion of this second barcoding step, cells are again collected from each well of the second plate, pooled together, and redistributed into a third multi-well device. Optionally, cells or nuclei are lysed and nucleic acids are purified in each well independently. PCR reagents are then added to the wells, including a thermostable polymerase, dNTPs, buffer, and two PCR primers. The first primer is specific to an adapter handle sequence (second primer binding) present in the primer used in the second strand synthesis step, and one or more second primers that have a region at their 3' end that is complementary to either a gene-specific sequence (e.g., a TCR constant region gene sequence) nested internally to the reverse primer used in the second step, or an adapter handle included in the reverse primer used in the second indexing step. One or both of these PCR primers may contain a barcode sequence (i.e., a third identifier tag) that, alone or in combination, provides a third cell-specific barcode sequence.

[0145] Optionally, the PCR primers may also include additional sequences for next generation sequencing, such as lead primer sequences, sequencing platform adaptor constructs such as p5, p7 sequences, etc. Alternatively, these sequencing platform adaptor construct sequences may be added in another round of PCR following the PCR reaction used to add the third identifier sequence (third cell-specific barcode sequence).

[0146] The resulting sequencing products are then sequenced using a next-generation sequencing platform to obtain gene sequence information and barcode information. The combination of barcodes from each indexing round identifies the cells or nuclei whose individual cell or nucleus sequences arose from the unique passage of the cell or nucleus through the first, second, and third unique wells of each respective multi-well device. That is, for example, the first (TSO) barcode, the second barcode (from second strand synthesis) and the third barcode from PCR provide each individual cell or nucleus with a unique cell-specific barcode that can track the gene-specific sequence. Thus, the gene or gene expression detected in each of the cells or nuclei of the original collection is determined.

[0147] Figure 6 provides a schematic diagram showing the structure of an NGS library product generated using an embodiment of the invention as described in Example 2. More specifically, T cell receptor genes are specifically targeted for analysis. As shown in Figure 6, read 1 provides the sequence of the targeted TCR gene. Read 2 also provides the sequence of the T cell receptor gene along with the first two index sequences, namely index 2 (IN2) from the second indexing step and index 1 (IN1) from the first indexing step. Index 3 is shown as provided by a combination of the i7 and i5 indexes added by PCR in the third indexing step.

[0148] Example 3: Embodiment of the present invention Example 3 is shown broadly in Figure 4 and is carried out in a similar manner to Example 1, except for the method of adding the 3' barcode in the second step, as described below. This example follows the same initial steps as Example 1, adding a first cell-specific barcode (i.e., a well-specific first identifier tag). The steps of this methodology are described below.

[0149] The cells or nuclei are fixed and permeabilized in an appropriate solution (e.g., 1% or 4% paraformaldehyde with NP40 or digitonin). The fixed cells or nuclei are aliquoted into the wells of a multi-well device (e.g., a 96-well or 384-well plate). Alternatively, the wells do not have to be physical, but may be composed of droplets, with the cells or nuclei being distributed into different droplets. The number of cells or nuclei per well or vessel may vary depending on the scale of the experiment the researcher wishes to perform. For example, 100 or 1,000 cells per well for each of the 96 or 384 wells of the plate. A reverse transcription mixture containing reverse transcriptase, oligo-dT, a template switch oligo (TSO) containing a first cell-specific barcode (i.e., a well-specific first identifier tag) and an adapter handle for PCR (i.e., a primer binding site), dNTPs, and buffer salts is added to each of the wells, and the RT reaction is allowed to proceed (e.g., at any suitable temperature, such as 37-50 °C, e.g., 42 °C, etc., for a time sufficient to complete the reaction, e.g., 60-90 min or more). This is shown in step A of Figure 4. The reaction is stopped, and the cells or nuclei are collected from each of the wells, pooled together, and then redistributed into the wells of a second multi-well device. Alternatively, the cells or nuclei may be redistributed into another set of droplets.

[0150] If desired, a lysis buffer is added to each of the wells to release the nucleic acid from each of the cells or nuclei. Optionally, the nucleic acid is purified independently in each well. Reagents effective to perform PCR are then added to the wells as illustrated in step B-PCR1 of FIG. 4. These may include a thermostable polymerase, dNTPs, a buffer, and two or more PCR primers. A first primer is specific to an adapter handle (primer binding site) sequence present in the TSO used in the reverse transcriptase step, and one or more second primers that have a region at their 3' end that is complementary to either a gene-specific sequence (e.g., a TCR constant region gene sequence), a polyA sequence, an adapter handle, or a random sequence. This is shown as step B in FIG. 4. In this step, all or a portion of a second cell-specific barcode sequence (second identifier) ​​is included in the primer that binds to the primer binding site in the TSO. In FIG. 4, this is shown as BC2A. After amplification, a hairpin adaptor containing all or a subportion of the second cell-specific barcode is added to the 3' end of the fragments generated in step B using a modified version of the ThruPLEX® Library Preparation Kit (Takara Bio USA Inc., San Jose, Calif.), i.e., using only a single adaptor, and an adaptor bearing the barcode is added to the 3' end of the fragments. This is detailed in step C of FIG. 4.

[0151] Optionally, tagmentation can be used instead of ThruPLEX® to add barcode adaptors to the ends of the fragments. After the addition of the adaptors, PCR is performed to amplify only the sequences with adaptors on the 3' end and add any additional sequences required for next generation sequences, such as, for example, Illumina P7 sequences. Optionally, for example, to reduce the total number of barcoded oligos required, the primer specific for the handle in the template switch oligo can include a sub-portion of the second cell-specific barcode (i.e., a second identifier tag). This sub-identifier (BC2A in FIG. 4) when combined with the sub-identifier provided by the tag in the 3' adaptor (BC2B in FIG. 4) provides the second cell-specific barcode sequence (i.e., the second identifier tag). It should be understood by one of skill in the art that either BC2A or BC2B can be used as the entire second identifier tag without the need to use the other tag, or a combination of BC2A and BC2B can be used to provide a combined second identifier.

[0152] The resulting sequencing products are then sequenced using a next-generation sequencing platform to obtain gene sequence information and barcode information (a combination of the first (TSO) barcode, the second barcode (from second strand synthesis), and the third barcode from PCR), providing each individual cell or nucleus with a unique cell-specific barcode that can track gene-specific sequences. Thus, the expression of the gene or genes detected in each of the cells or nuclei of the original collection is determined.

[0153] As illustrated in Figures 2, 3, and 4, barcodes BC2A and 2B can be combined to be computationally a single unique secondary barcode that uniquely defines the wells of the second pooling step. BC1 and BC2 combine to uniquely define cells from the original pool.

[0154] Example 4 Two rounds of barcoding of TCR beta chains from a mixture of Jurkat and CCRF-CEM cells Figures 8A and 8B illustrate the two-round barcoding protocol used to prepare the TCR sequencing library. As shown in Figure 8A, Jurkat and CCRF-CEM cells were fixed by incubating with 4 volumes of cold methanol at -20°C for 30 minutes (Figure 8A, step B). Fixed cells were removed from the -20°C freezer and rehydrated on ice with 500 ul of rehydration buffer containing PBS buffer, BSA, RNase Inhibitor, and DTT after removal of the methanol. 1,000 fixed Jurkat and CCRF-CEM cells were allocated into each of three tubes and two tubes with PBS as a negative control (Figure 8B). A reverse transcription mix containing reverse transcriptase, RNase inhibitor, poly-dT oligo, template switch oligo (TSO) containing the first tube-specific barcode (BC1) and Illumina RP1 sequence, dNTPs, and RT buffer was added to each tube, and the RT reaction was carried out at 42° C. for 90 min (FIG. 8A, step C). Cells were collected from each tube and pooled together (FIG. 8A, step D). After centrifugation, the supernatant was discarded and the cells were resuspended in PBS buffer. The resuspended cells were redistributed into a new set of eight tubes (FIG. 8A, step E, and FIG. 8B).

[0155] A PCR1 mixture containing DNA polymerase, a PCR primer containing the Illumina RP1 sequence, a primer that specifically hybridizes to the constant region of the T cell antigen receptor (TCR) beta chain (TCRb PCR1 primer), dNTPs, and PCR buffer was added to the eight tubes containing the resuspended cells. PCR1 was then performed in 40 μl (FIG. 8A, step F). The TCRb PCR1 primer is a chimeric DNA / RNA oligonucleotide that functions as a PCR primer in the absence of RNases, but can be inactivated after PCR by digestion with various RNases (e.g., RNase H, RNase A, etc.), as described, for example, in U.S. Patent Application Serial No. 16 / 603,788 (Attorney Docket No. CLON-169), published as US2020-0332341A1 (the disclosure of which is incorporated herein by reference).

[0156] A PCR2 mix containing DNA polymerase; RNaseH; BC2a(i5) primer containing Illumina RP1 sequence, BC2a(i5) and P5 adaptor sequence; BC2b(i7) primer containing Illumina RP2 sequence, BC2b(i7) and P7 adaptor sequence; TCRb PCR2 primer (hybridizes to the TCRb constant region at a point internal to the TCRb PCR1 primer and further contains the Illumina RP2 sequence); dNTPs; and PCR buffer was added directly to the eight tubes containing the PCR1 reaction products. PCR2 was then performed on 70 ul (Figure 8A, step H). The resulting TCRb library contains all molecules marked with both the first and second round barcodes (BC1, BC2a(i5) and BC2b(i7)).

[0157] The eight barcoded TCRb libraries were purified with magnetic beads and quantified by Qubit, Bioanalyzer High sensitivity kit (one of the eight libraries is shown in Figure 8C) and qPCR. They were then pooled together and loaded into a NextSeq sequencer (Illumina Inc., San Diego, CA) for paired-end sequencing (2 × 151PE). The resulting sequencing reads were demultiplexed with BC1, BC2a (i5) and BC2b (i7) and analyzed by Cogent AP software (Takara Bio USA, Inc., San Jose CA). All 64 expected barcode combinations (8 first-round barcodes × 8 second-round barcodes) were detected and the results were plotted based on the number of reads of the detected Jurkat clonotype (amino acid sequence: CASSFSTCSANYGYTF) and CCRF clonotype (amino acid sequence: CASSLGTDTQYF) (Figure 8D). As expected, most reads were assigned to Jurkat or CCRF clonotypes, confirming that the combined barcoding strategy, including the use of first-round barcodes from the TSO, was working as expected.

[0158] However, an alternative barcoding strategy is also envisioned, in which the forward primer for PCR1 contains the partial second round barcode BC2a, the P5 sequence, and the Illumina RP1 sequence, as shown in FIG.

[0159] Example 5 Three rounds of barcoding of TCR alpha and beta chains from PBMC RNA Sequencing libraries were prepared using the three-round barcoding protocol diagrammed in Figure 10A. PBMC RNA (Takara Bio USA Inc.) was diluted to 5ng / ul and 2ul (10ng) was dispensed into Eppendorf tubes. As a negative control, 2ul of RNase-free water was dispensed into another tube. Reverse transcription mix containing reverse transcriptase, RNase inhibitor, poly-dT oligo, template switch oligo (TSO) containing the first barcode (BC1) and primer binding site for second strand synthesis (second SS handle), dNTPs, and RT buffer was added to each tube and RT reactions were performed in 20ul at 42°C for 90 minutes followed by incubation at 70°C for 10 minutes (Figure 10A, step C). The TSO used in this reaction is a chimeric DNA / RNA oligonucleotide that functions as a template-switching oligo in the absence of RNases, but can be inactivated after use by digestion with various RNases (e.g., RNase H, RNase A, etc.), as described, for example, in U.S. Patent Application Serial No. 16 / 603,788 (Attorney Docket No. CLON-169), published as US2020-0332341A1 (the disclosure of which is incorporated herein by reference). The RT product containing BC1 was purified by magnetic beads and eluted in 13 ul of elution buffer.

[0160] Second strand synthesis mixture (2nd SS) containing reverse transcriptase; RNaseH; a sequence hybridizing to the TSO primer binding sequence, a 2nd SS oligo containing BC2 and Illumina RP2 sequence; dNTPs; and 2nd SS reaction buffer was added to the clean tube containing 13 ul of purified RT product. The 2nd SS reaction was then carried out at 42°C for 10 min in 20 ul, followed by incubation at 70°C for 10 min (Figure 10A, step F). The 2nd SS product with BC1 and BC2 was purified by magnetic beads and eluted in 12 ul of elution buffer.

[0161] A PCR1 mix containing DNA polymerase; BC3b(i7) primer containing Illumina RP2 sequence, BC3b(i7) and P7 adapter sequence; TCRa PCR1 primer that specifically hybridizes to the constant region of the T cell antigen receptor (TCR) alpha chain; TCRb PCR1 primer that specifically hybridizes to the constant region of the T cell antigen receptor (TCR) beta chain; dNTPs; and PCR buffer was added to a clean tube containing 10 ul of the purified 2nd SS product, and then PCR1 was performed with 40 ul (Figure 10A, step I). Both the TCRa PCR1 primer and the TCRb PCR1 primer described above are chimeric DNA / RNA oligonucleotides that can function as PCR primers in the absence of RNases, but can be inactivated after use by digestion with various RNases (e.g., RNase H, RNase A, etc.), as described, for example, in U.S. Patent Application Serial No. 16 / 603,788, published as US2020-0332341A1 (Attorney Docket No. CLON-169), the disclosure of which is incorporated herein by reference.

[0162] Following PCR1, a PCR2 mix containing DNA polymerase; RNaseH; BC3a(i5) primer containing Illumina RP1 sequence, BC3a(i5) and P5 adaptor sequence; TCRb PCR2 primer hybridizing to the TCRa constant region at an internal point relative to the TCRa PCR1 primer and further containing Illumina RP1 sequence; TCRb PCR2 primer hybridizing to the TCRb constant region at an internal point relative to the TCRb PCR1 primer and further containing Illumina RP1 sequence; dNTPs; and PCR buffer was added directly to the PCR1 tube. PCR2 was then performed in 70 ul (Figure 10A, step K). After PCR2, the TCR library containing molecules marked with the first, second and third round barcodes (BC1, BC2, BC3a(i5) and BC3b(i7)) was purified by magnetic beads and quantified by Qubit and Bioanalyzer High sensitivity kit. Libraries derived only from 10 ng of PBMC RNA were adapted to have the expected size (approximately 650 bp) (Figure 10 panel B).

[0163] The libraries were loaded onto an Illumina MiSeq (Illumina Inc, San Diego, CA) for paired-end sequencing (2x151PE), and the resulting sequencing data was analyzed using Cogent AP software (Takara Bio USA, Inc. San Jose CA). 410,696 reads were obtained after demultiplexing, and 286,070 reads (representing a mapping rate of 70%) were mapped to TCRa and TCRb. The number of clonotypes detected was 93 (TCRa) and 890 (TCRb) (Figure 10 panel C). This result demonstrates that the three-round combinatorial barcoding strategy using TSO to provide the first round barcode (first identifier) ​​and second strand synthesis to provide the second round barcode (second identifier) ​​works as expected.

[0164] Note that an alternative barcoding strategy is also envisaged, in which the forward primer for PCR1 does not contain a barcode sequence, as shown in FIG.

[0165] Example 6. Combined targeted sequencing and two rounds of barcoding for 5' differential expression (5'DE) Sequencing libraries were generated using the protocol illustrated in FIG. 12A. K562 and 3T3 cells were fixed using 1% paraformaldehyde (PFA) and permeabilized with 0.01% digitonin (FIG. 12A, step B). After washing, cells were aliquoted into 39 wells of a 96-well plate (8 wells for K562 cells, 8 wells for 3T3 cells, and 23 wells for a mixture of K562 and 3T3 cells) such that each well contained approximately 1,000 cells. Reverse transcription mix was added to each well, including reverse transcriptase; RNase inhibitor; RT oligo containing a poly-dT sequence and a PCR handle sequence; template switch oligo (TSO) containing a first well-specific barcode (BC1) and a primer binding site for PCR; dNTPs; and RT buffer, and the RT reaction was carried out at 42° C. for 90 minutes (FIG. 12A, step C). Cells from each of the wells were then collected and pooled together. After centrifugation, the supernatant was discarded and the cells were resuspended in PBS buffer.

[0166] The resuspended cells were redistributed into 1,296 wells of an ICELL8 nanowell chip (FIG. 12A, step E) using an ICELL8 instrument (Takara Bio USA, Inc., San Jose CA). Two forward PCR primers containing one of a pair of partial second-round well-specific barcodes (either BC2a or BC2b) were sequentially distributed onto the chip, along with a reverse PCR primer that hybridizes to the PCR handle sequence from the RT oligo, and PCR reagents containing DNA polymerase, dNTPs, and PCR buffer. The two forward primers were added such that one defines a specific row of wells on the chip and the other defines a specific column of wells. Thus, in combination, they define a unique well location. The first forward PCR primer contains a sequence that can hybridize to the PCR handle provided by the TSO, BC2a, and the Illumina RP2 sequence. The second forward PCR primer contains the Illumina RP2 sequence, BC2b, and the P7 sequence. Thus, combining these primers allows for a "step-out" PCR reaction that ultimately generates a PCR product that contains sequences from both primers in combination, as shown in Figure 12A, Step F.

[0167] After completion of this second barcoding step, the barcoded full-length cDNA was extracted from the ICELL8 chip by centrifugation and purified with magnetic beads. After quantification with Qubit and Bioanalyzer High sensitivity kit, the cDNA was used to prepare sequencing libraries for differential gene expression analysis (5'DE) using the Illumina Nextera XT kit, followed by PCR using P7 primers and Illumina P5 index primers (Figure 12B). After purification and quantification, the final 5'DE library was loaded onto an Illumina NextSeq for paired-end sequencing (2x75PE). The resulting sequencing reads were demultiplexed and analyzed using Cogent AP software (Takara Bio USA, Inc., San Jose CA).

[0168] After demultiplexing the sequencing reads using BC1, BC2a, BC2b (i7) and i5 indexes using Cogent AP software, 1310 cells with over 10,000 reads / cell were identified (Figure 12C). Data from these cells were used for downstream analysis (as shown in Figures 12D-12F). Notably, the L-plot analysis shown in Figure 12F clearly showed a high mapping rate of the data to either the human genome (hg38; representative of K562 cells) or the mouse genome (mm10; representative of 3T3 cells), with a very low doublet rate. This demonstrated that this combinatorial barcoding strategy was capable of generating single-cell data.

[0169] Example 7 Two rounds of barcoding for targeted sequencing and 5' differential expression (5'DE) of combined TCR chains using PBMC RNA PBMC RNA (Takara Bio USA Inc., San Jose CA) was diluted to 5.0 ng / ul and 2 ul (10 ng) was dispensed into Eppendorf tubes. Reverse transcription mix containing reverse transcriptase; RNase inhibitor; RT oligo containing poly-dT sequence and PCR handle sequence; template switch oligo (TSO) containing first well-specific barcode (BC1) and primer binding site for PCR; dNTPs; and RT buffer was added to the tube and RT reaction was carried out for 20 ul at 42°C for 90 min followed by incubation at 70°C for 10 min (Figure 12A, step C). RT product containing BC1 was purified by magnetic beads and eluted in 12 ul elution buffer.

[0170] Two forward PCR primers containing one of the partial second round well-specific barcode (either BC2a or BC2b) pairs were added to the tube along with a reverse PCR primer that hybridizes to the PCR handle sequence from the RT oligo, and PCR reagents including DNA polymerase, dNTPs, and PCR buffer. The two forward primers were added such that one provides the partial second round barcode sequence BC2a and the other provides the partial second round barcode sequence BC2b. Thus, in combination, they define a unique second round barcode. The first forward PCR primer contains a sequence that can hybridize to the PCR handle provided by the TSO, BC2a, and an Illumina RP2 sequence. The second forward PCR primer contains an Illumina RP2 sequence, BC2b, and a P7 sequence. Thus, as shown in Figure 12A, step F, these primers together allow for a "step-out" PCR reaction that ultimately generates a PCR product that contains sequences from both primers in combination.

[0171] After completing this second barcoding step by PCR, the barcoded full-length cDNA was purified with magnetic beads. After quantification by Qubit and Bioanalyzer High sensitivity kit (Figure 13B), the cDNA was aliquoted into three tubes as follows: 1 tube for TCRa, Tube 2 for TCRb, and Tube 3 for TCRa and b.

[0172] PCR1 mix containing DNA polymerase, P7 primer, dNTPs, and PCR buffer was added to each of the three tubes. Then, TCRa PCR1 primer, which specifically hybridizes to the constant region of the T cell antigen receptor (TCR) alpha chain, was added to tube 1. TCRb PCR1 primer, which specifically hybridizes to the constant region of the T cell antigen receptor (TCR) beta chain, was added to tube 2, and both TCRa and TCRb PCR1 primers were added to tube 3. PCR1 was then run with 40 ul (Figure 13A). Both the TCRa PCR1 primer and the TCRb PCR1 primer described above are chimeric DNA / RNA oligonucleotides that can function as PCR primers in the absence of RNases, but can be inactivated after use by digestion with various RNases (e.g., RNase H, RNase A, etc.), as described, for example, in U.S. Patent Application Serial No. 16 / 603,788, published as US2020-0332341A1 (Attorney Docket No. CLON-169), the disclosure of which is incorporated herein by reference.

[0173] Following PCR1, a PCR2 mix containing DNA polymerase; RNaseH; a P5 index primer with an i5 index containing an Illumina RP1 sequence, an i5 index, and a P5 adapter sequence; dNTPs; and PCR buffer was added to each of the three tubes along with either a TCRa PCR2 primer that specifically hybridizes the constant region of the T cell antigen receptor (TCR) alpha chain (tube 1), a TCRb PCR2 primer that specifically hybridizes the constant region of the T cell antigen receptor (TCR) beta chain (tube 2), or both the TCRa and TCRb PCR2 primers (tube 3). Each of the TCRa and b PCR2 primers also contain the RP1 sequence. PCR2 was then run with 70 ul (Figure 13A).

[0174] After PCR2, the TCR library with all three barcode sequences of BC1, BC2a, BC2b (i7) and TCR specific index (i5) was purified by magnetic beads and quantified by Qubit, Bioanalyzer High sensitivity kit (Figure 13C) and qPCR. The library was loaded into an Illumina MiniSeq sequencer for paired-end sequencing (2x151PE), and the resulting sequencing data was analyzed with Cogent AP software (Takara Bio USA, Inc., San Jose CA). A summary of the sequencing results is provided in Table 1 below: [Table 1]

[0175] The results demonstrate that this combination barcoding strategy works.

[0176] Example 8. Combined targeted sequencing and three rounds of barcoding for 5' differential expression (5'DE) A protocol for generating targeted sequencing and 5' differential expression libraries using three rounds of barcoding is shown in Figure 14. In this example, cells are fixed and distributed across the wells of a plate (e.g., a 96-well plate). A reverse transcription mixture containing reverse transcriptase, RNase inhibitor, poly-dT oligo, template switch oligo (TSO) containing a first barcode (BC1) and a primer binding site (second SS handle) for second strand synthesis, dNTPs, and RT buffer is added to each well, and the RT reaction is performed (Figure 14, step C). The TSO used in this reaction is a chimeric DNA / RNA oligonucleotide that functions as a template switching oligo in the absence of RNase, but can be inactivated after use by digestion with various RNases (e.g., RNase H, RNase A, etc.), as described, for example, in U.S. Patent Application Serial No. 16 / 603,788 (Attorney Docket No. CLON-169), published as US2020-0332341A1 (the disclosure of which is incorporated herein by reference). The cells from each well containing BC1 are then pooled (Figure 14, step D). The pooled cells are then redistributed into a second set of wells in a new multiwell plate or nanowell chip (Figure 14, step E).

[0177] A second strand synthesis mix (2nd SS) containing reverse transcriptase; RNaseH; a sequence hybridizing to the TSO primer binding sequence, a 2nd SS oligo containing BC2 and a PCR handle sequence; dNTPs; and a 2nd SS reaction buffer is then added to each well. A 2nd SS reaction is then performed (Figure 14, step F). The cells containing the 2nd SS product containing BC1 and BC2 are then pooled (Figure 14, step G). The pooled cells are then redistributed to a third set of wells in a new multiwell plate or nanowell chip (Figure 14, step H).

[0178] Two forward PCR primers containing one of the partial third round well-specific barcode (either BC3a or BC3b) pairs are added to each well along with a reverse PCR primer that hybridizes to the PCR handle sequence from the RT oligo, and PCR reagents containing DNA polymerase, dNTPs, and PCR buffer. The two forward primers are added such that one provides the partial third round barcode sequence BC3a and the other provides the partial third round barcode sequence BC3b. Thus, in combination, they define a unique third round barcode. The first forward PCR primer contains a sequence that can hybridize to the PCR handle provided by the second strand synthesis primer from the second barcoding round, BC3a, and an Illumina RP2 sequence. The second forward PCR primer contains an Illumina RP2 sequence, BC3b, and a P7 sequence. Thus, as shown in Figure 14, Step I, these primers together allow for a "step-out" PCR reaction that ultimately generates a PCR product that contains sequences from both primers in combination.

[0179] After completion of this third barcoding step by PCR, the barcoded full-length cDNA can be converted into a final 5'DE and / or TCR or other gene specific library using a process similar to that described in either Example 6 (for 5'DE using Nextera (Illumina, Inc., San Diego CA)) or Example 7 (2-round PCR for TCR-specific library generation). The steps for this and the structure of the final library generated are shown in Figure 15. Figure 15 panel A shows the steps for generating a 5'DE library using tagmentation (e.g., using Nextera (Illumina, Inc., San Diego CA)). In other embodiments, combined fragmentation and ligation of hairpin adaptors can be used as described in the SMART-Seq Library Prep Kit (catalog number 634764, Takara Bio USA, Inc. San Jose CA). Figure 15 panel B shows the steps for generating a TCR library after the third barcoding round.

[0180] Example 9 Analysis of Template-Switching Oligos (TSOs) with Different Indexes Using Purified RNA The performance of TSOs with different unique 8 nt indexes was tested in reverse transcription (RT) reactions. 10 ng of purified K562 RNA was mixed with 4 μl of RT buffer (250 mM Tris, 375 mM KCl, 30 mM MgCl2), 1 μl of random hexamers, 1 μl of nuclease-free water, and fragmented by heating at 85° C. for 6 minutes, then immediately chilled on ice. RT Mastermix containing 4.5 ul of TSO buffer, 0.5 ul of RNase Inhibitor, 1 ul of reverse transcriptase (200 u / ul), 1 ul of TSO (50 μM) with an 8 nt first identifier (i.e., first index), and 6 ul of RNase-free water was added to the fragmented RNA. Each well of a 96-well plate contained a TSO with a unique first identifier. The RT reaction was carried out at 25°C for 10 min, 42°C for 90 min, then 72°C for 5 min. After the RT reaction, PCR Mastermix containing 25ul 2X CB buffer, 1ul DNA polymerase, 1ul 5' PCR primer, and 1ul 3' PCR primer was added to amplify the RT product with the following PCR program: 94°C, 1 min; 10 cycles of 98°C, 15 sec, 55°C, 15 sec, 68°C, 30 sec; 68°C, 2 min; hold at 4°C. The PCR products were purified by magnetic beads, and the concentrations of the PCR products were measured by Qubit.

[0181] As shown in Figure 16, all tested TSOs with 8 nt first identifiers produced good library yields. Without TSO, the RT reaction had very low yields. This result indicates that the addition of the first identifier using TSO during the RT step was successful using purified RNA.

[0182] The length of the first identifier can vary between 6 and 12 nucleotides (nt). 2+The concentration can vary between 2 and 12 mM. The length of the random primer can vary between 6 and 15 nt. The final TSO concentration in the RT reaction can vary between 0.5 and 5 μM. The final random primer concentration in the RT reaction can vary between 0.5 and 5 μM. The RNA fragmentation temperature can vary between 65°C and 95°C, and the incubation time can vary between 1 and 30 minutes. The RT reaction can be a constant temperature incubation or a temperature gradient with or without thermal cycling.

[0183] Example 10. Analysis of Combinatorial Indexing in Single Cells Cells or nuclei were fixed in an appropriate fixative solution (e.g., 1-4% paraformaldehyde, glyoxal, DSP, DST, methanol, etc.). Fixed cells or nuclei were aliquoted into wells of a multi-well device (e.g., 96-well or 384-well plates, nanowell ICELL8 chips, etc.). Cellular RNA was fragmented, and then a reverse transcription Mastermix containing cell permeabilization reagent (e.g., 0.01-0.5% digitonin, saponin, Tween20, Triton X-100, NP40, etc.) was added to the heat-treated cells. A first identifier was added during the in situ reverse transcription (RT) reaction by using template-switching oligos (TSOs), each of which carries a unique first identifier. The cells containing the cDNA with the first identifier were then pooled and then split again into multiple partitions (e.g., 96-well or 384-well plates, or 5184 nanowell ICELL8 chips (Takara Bio USA, Inc. San Jose CA)) such that each second partition contained multiple cells with a different first identifier. A PCR Mastermix with primers carrying a unique second identifier was then added to each partition, and a PCR reaction was performed to incorporate the second identifier into the final library DNA. If necessary, the cells can undergo additional pool splitting rounds, and more identifiers can be added by amplification or ligation. Multiple rounds of identifier addition steps were performed manually or by automation, such as a robotic liquid handler. The final library DNA from each individual cell has a unique combination of identifiers. rRNA was then depleted from the library, and after cleanup and quantification, the library was sequenced on a sequencer (e.g., Miseq, NextSeq, Novaseq, etc., all manufactured by Illumina Inc., San Diego CA).

[0184] In this example, cells (K562 (human):3T3 (mouse) in a 1:1 ratio) were fixed with 1% paraformaldehyde and aliquoted across the wells of a 96-well plate such that each well contained approximately 2000 cells per well. RNA fragmentation was performed by mixing 5 μl of fixed cells with 4 μl of RT buffer (250 mM Tris, 375 mM KCl, 30 mM MgCl2) and 1 μl of 12 μM random hexamers, then heating the cells to 85° C. for 6 minutes. The cells were then immediately cooled on ice. An RT mix containing 4.5 ul of TSO buffer, 1 ul of digitonin (0.2%), 0.5 ul of RNase Inhibitor, 1 ul of reverse transcriptase (200 U / ul), and 1 ul of RNase-free water was prepared and added to each well of the plate. The RT reaction was then carried out by adding 1 ul of TSO (50 uM) bearing a unique first identifier to each well of the 96-well plate and incubating the PATE at 25° C. for 10 minutes and then at 42° C. for 90 minutes. After the RT reaction was completed, cells from all wells of the 96-well plate were pooled together, washed once with PBS, and the cells were resuspended leaving 30ul of liquid, then distributed across the nanowells of the ICELL8 chip using an ICELL8 instrument (Takara Bio USA, Inc., San Jose CA). The i5, i7 index, used as the second identifier in this experiment, and PCR mix (SeqAmp DNA polymerase and 2X CB buffer, both supplied by Takara Bio USA, Inc., San Jose CA) were then distributed to the ICELL8 chip and mixed with the cells. PCR was performed on-chip with the following program: 94°C, 1 min; 10 cycles of 100°C, 15 sec, 49.3°C, 5 sec, 54.5°C, 10 sec, 72.2°C, 9 sec, 67.9°C, 31 sec; 67.9°C, 2 min; hold at 4°C. After the PCR reaction, the libraries were pooled and cleaned up by magnetic beads.The beads were eluted with ZapR mix (Takara Bio USA, Inc. San Jose CA) containing 2.2 μl 10× ZapR buffer, 1.5 μl scZapR, 1.5 μl heated probe and 16.8 μl nuclease-free water, and the ZapR reaction was carried out at 37° C. for 1 hour and 72° C. for 10 minutes to remove rRNA from the library. After ZapR was completed, a second PCR reaction was performed to amplify the library by adding 80 ul of PCR mix (2 ul SeqAmp DNA polymerase, 2 ul PCR2 primer, 50 ul 2× CB buffer, and 26 ul nuclease-free water) to each tube containing 20 ul of ZapR product with the following program: 94° C., 1 min; 5 cycles of 98° C., 15 sec, 55° C., 15 sec, 68° C., 30 sec; hold at 4° C. After the second PCR reaction, the products were purified by magnetic beads and quantified by Qubit, BioAnalyzer and qPCR. Based on the quantification, the library DNA was diluted to 4 nM. 5 ul of 4 nM library was mixed with 5 ul of freshly made 0.2 N NaOH and incubated at room temperature for 5 minutes. Next, 5 μl of 200 mM Tris-HCl (pH 7) was added, followed by 985 μl of HT buffer (Illumina Inc. San Diego CA), resulting in a 20 pM denatured library. This was then diluted to 1.5 pM as follows: The denatured library solution (97 μl) and Prechilled HT1 (1203 μl) were loaded onto a Nextseq cartridge (Illumina Inc. San Diego CA) for sequencing.

[0185] As shown in Table 2, 93.4% of the sequencing reads were successfully barcoded, and only 6.6% of the total reads were pending. The demultiplexed reads were used to create a "knee plot" to determine the number of cells passing QC that could be identified. As shown in Figure 17, 1133 cells were successfully barcoded and passed the QC threshold (20,000 reads / cell). This demonstrates that combinatorial indexing was achieved by adding a first identifier using template switching with TSO and a second identifier using PCR. In this case, the second identifier is in the form of 2a+2b, i5 and i7, which each define either a row or a column of the ICELL nanowell chip, but the combination is unique to a particular well. The combination of the incorporated first and second identifiers is unique to each individual single cell from the original pool of fixed cells. The Cogent NGS Analysis Pipeline (Takara Bio USA, Inc. San Jose CA) was used to analyze the sequencing reads and map them to both the human and mouse genomes. The mapping results were used to generate an "L-plot" that was used to determine the percentage of cells captured individually, as well as the presence of cell doublets. As shown in FIG. 18, the x-axis of the L-plot shows the reads mapped to the human genome, and the y-axis shows the reads mapped to the mouse genome. Human and mouse cells were well separated on the L-plot. The doublet ratio was calculated to be 9.8%, close to the expected ratio of 10.5% that was expected based on the number of indexes used and the total number of cells assayed. This demonstrates that there was minimal cell-to-cell crosstalk during the combined indexing workflow, and thus single cells can be individually identified using this method. As shown in Table 3, the sequencing data showed overall good mapping metrics with good exon and intron ratios, as well as low intergenic / mitochondrial / ribosomal ratios. 5896 genes were detected at an average sequencing depth of 148,000 reads / cell.These data demonstrate that the invention as implemented according to this exemplary embodiment can analyze single-cell total RNA-seq in a high-throughput manner with good performance. [Table 2] [Table 3]

[0186] Example 11 Analysis of combined indexing at the single cell level with high concentrations of PFA and digitonin In this example, 4 million cells (K562:3T3 at a 1:1 ratio) were centrifuged at 300g for 3 minutes. The pellet was resuspended in 1 ml of 4% PFA and incubated on ice for 15 minutes to fix the cells. The cells were then pelleted by centrifugation at 500g for 5 minutes. The cell pellet was resuspended in 1 ml of 3 mM glycine (pH 7.5) and incubated on ice for 5 minutes to quench the fixation process. The cells were pelleted again by centrifugation at 500g for 5 minutes to remove the glycine solution, and then resuspended in PBS containing 1% second diluent and 1% RNase inhibitor. 9 μl of fixed cells were mixed with 4 μl of RT buffer (250 mM Tris, 375 mM KCl, 30 mM MgCl2) and incubated at 85° C. for 6 minutes to perform RNA fragmentation. Next, 1ul of 1.4% digitonin was added to the heated cells and the mixture was incubated at room temperature for 5 minutes to permeabilize the cells. The final digitonin concentration was 0.1%. The RT mixture (4.5ul of scTSO mixture, 0.5ul of RNase inhibitor, 1ul of reverse transcriptase (200u / ul), 1ul of 12uM random primers) was added to each well of the 96-well plate containing the permeabilized cells and the plate was incubated at 42°C for 90 minutes to allow the RT reaction. After RT, the cells from all wells of the 96-well plate were pooled together and washed once with PBS with 0.04% BSA, the cells were resuspended leaving 25ul of liquid, and then distributed into the nanowells of an ICELL8 chip using an ICELL8 instrument (Takara Bio USA, Inc. San Jose CA). Then, the i5, i7 indexes and PCR mix (SeqAmp DNA polymerase and 2X CB buffer - both from Takara Bio USA, Inc. San Jose CA) were dispensed into the ICELL8 chip and mixed with the cells. PCR was performed on-chip with the following program: 72.1°C, 3 min; 98.2°C, 18 sec; 96.5°C, 42 sec; 10 cycles of 100°C, 10 sec, 54.4°C, 5 sec, 59.6°C, 10 sec, 72.2°C, 9 sec, 67.9°C, 1 min 51 sec; hold at 4°C. After the PCR reaction, the libraries were pooled and cleaned up using magnetic beads.The beads were eluted with ZapR mix (Takara Bio USA, Inc. San Jose CA) containing 2.2 μl 10× ZapR buffer, 1.5 μl scZapR, 1.5 μl heated probe mix, and 16.8 μl nuclease-free water. The ZapR reaction (Takara Bio USA Inc. San Jose CA) was then carried out at 37° C. for 1 hour, followed by incubation at 72° C. for 10 minutes to remove rRNA from the library. After the ZapR reaction was completed, a second PCR reaction was performed to amplify the library by adding 80ul of PCR mix (2ul SeqAmp DNA polymerase, 2ul PCR2 primer, 50ul 2X CB buffer, and 26ul nuclease-free water) to each tube containing 20ul of ZapR product with the following program: 94°C, 1 min; 5 cycles of 98°C, 15s, 55°C, 15s, 68°C, 30s; hold at 4°C. After the PCR reaction was completed, the products were purified by magnetic beads and quantified by Qubit, BioAnalyzer and qPCR. Based on the quantification, the library DNA was diluted to 4nM. 5ul of the 4nM library was mixed with 5ul of freshly made 0.2N NaOH and incubated at room temperature for 5 minutes. Next, 5 μl of 200 mM Tris-HCl (pH 7) was added, followed by 985 μl of HT buffer (Illumina Inc. San Diego CA), resulting in 20 pM of denatured library, which was then diluted to 1.7 pM and loaded onto a Nextseq cartridge (Illumina Inc., San Diego CA) for sequencing.

[0187] After sequencing, the sequencing reads mapped to both human and mouse genomes were analyzed using Cogent NGS analysis Pipeline (Takara Bio USA, Inc. San Jose CA). The mapping results were used to generate an L-plot. As shown in FIG. 19, the x-axis showed the reads mapping to the human genome, and the y-axis showed the reads mapping to the mouse genome. The results showed that human and mouse cells were well separated on the L-plot. The doublet ratio was 5.8%, which was close to the calculated expected ratio of 5%. This demonstrated that there was minimal cell-cell crosstalk during the combined indexing workflow with 4% PFA and 0.1% digitonin.

[0188] Example 12 Testing different cell fixation solutions In this example, K562 cells were used to test different fixation solutions. Two million cells were first washed with PBS and then divided into five tubes, labeled 1-5. The cells in each tube were pelleted by centrifugation at 200g for 5 minutes. In each tube (1-5), the cell pellet was thoroughly resuspended in 0.5 mL of one of the following: 4% PFA, 1% PFA, 0.5% PFA, 0.25% PFA, or PBS. The cells were then incubated on ice for 10 minutes. 25 μl of 2% digitonin was then added to the cells in each tube and incubated on ice for 3 minutes to permeabilize the cells. After permeabilization, 2 mL of quench solution (1 M Tris-Cl (pH 8), 1% RNase inhibitor, 1% BSA) was added to tubes 1-4 to quench the cell fixation. 2 mL of PBS solution (PBS, 1% RNase inhibitor, 1% BSA) was added to tube 5 as a control. The cells were then pelleted by centrifugation at 200 g for 10 min at 4 °C and resuspended in 200 μl of resuspension solution (PBS, 1% RNase inhibitor, 1% second diluent, Takara Bio USA Inc. San Jose CA). 10 μl of cells in each tube were mixed with 10 μl of trypan blue and checked under a microscope. As shown in Table 4, a wide range of PFA concentrations (0.25%-4%) showed very good cell recovery without forming large cell clumps after fixation and cell permeabilization. The control cells without cell fixation and with digitonin treatment formed obvious large cell clumps with very few single cells. This indicated that a wide range of PFA concentrations could be used for cell fixation for single cell studies. [Table 4]

[0189] Example 13 Testing different cell permeabilization conditions by titration of digitonin In this example, K562 cells were used to test different permeabilization conditions. Two million cells were first washed with PBS and then pelleted by centrifugation at 200g for 5 minutes. The cell pellet was thoroughly resuspended in 0.5mL of 1% PFA for fixation. The cells were then incubated on ice for 10 minutes. Then, 2mL of quenching solution (1M Tris-Cl (pH8), 1% RNase inhibitor, 1% BSA) was added to quench the cell fixation. The cells were then pelleted by centrifugation at 200g for 10 minutes at 4°C and resuspended in 200μl of resuspension solution (PBS, 1% RNase inhibitor, 1% second diluent, Takara Bio USA Inc. San Jose CA). The cells were then counted and diluted in resuspension solution at a concentration of 200,000 cells / mL. 10 μl of cells in each tube were mixed with 10 μl of trypan blue and 2 μl of digitonin at two different concentrations and checked under a microscope. As shown in Table 5, cells without digitonin (sample ID3) were barely stained blue by trypan blue, indicating that the cell membrane fixed by 1% PFA was not permeable when digitonin was not added. When digitonin (0.1% or 0.01%) was added, all cells were stained blue by trypan blue (sample IDs 1 and 2), indicating that a wide range of digitonin concentrations permeable the cell membrane. [Table 5]

[0190] 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 that, in light of the teachings of this invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0191] Thus, the above merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art can devise various arrangements that embody the principles of the present invention and are included within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to further the art, and should not be construed as limitations to the examples and conditions specifically recited as such. Furthermore, all statements herein that describe principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, 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.

[0192] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 293,589, filed December 23, 2021, the disclosure of which is incorporated herein by reference.

Claims

1. 1. A method for preparing a multiple cell source distinguishable collection of nucleic acids derived from an initial multiple cell source, the method comprising: (a) providing a first set of cell source sub-portions, each sub-portion including a plurality of cell sources from the initial plurality of cell sources; (b) generating first identifier-tagged nucleic acids in the plurality of cell sources in each subportion of the first set using a template-switching-mediated reaction with a template switch oligonucleotide comprising a first identifier, wherein the first identifiers of the template switch oligonucleotides used in different subportions of the first set are identical within a given subportion but different among the different subportions; (c) pooling the cell sources of the subportions produced in step b) to generate a first cell source pool comprising a first identifier-tagged nucleic acid; (d) allocating the first cell source pool into a second set of subportions, each subportion including a plurality of cell sources including a first identifier-tagged nucleic acid; (e) producing cell-source-distinguishable nucleic acids from the plurality of cell sources in each subportion of the second set that include both a first identifier and a second identifier, wherein the second identifier for each subportion of the second set is identical within a given subportion but different between different subportions; Thereby, a plurality of cell-source-identifiable nucleic acid collections are prepared from the initial plurality of cell sources, wherein the nucleic acids of each cell-source-identifiable nucleic acid collection comprise a unique combination of first and second identifiers that identify the cell source of the nucleic acid.

2. The method of claim 1 , wherein the cellular source is a cell.

3. The method of claim 1 , wherein the cellular source is a nucleus.

4. The method according to any one of claims 1 to 3, wherein the cell source is permeabilized.

5. 10. A method according to any one of the preceding claims, wherein the first set of sub-portions comprises between 2 and 25,000 sub-portions.

6. 2. The method of any one of the preceding claims, wherein the template switch oligonucleotide further comprises a unique molecular identifier.

7. 2. The method of any one of the preceding claims, wherein the template-switching mediated reaction uses a ribonucleic acid template.

8. The method of claim 7, wherein the ribonucleic acid template is mRNA.

9. 9. The method of claim 8, wherein the template switching reaction uses oligo-dT primers, random primers, quasi-random primers, or gene-specific primers.

10. The method of any one of claims 1 to 6, wherein the template-switching mediated reaction uses deoxyribonucleic acid.

11. The method of claim 10, wherein the template switching reaction uses random primers, quasi-random primers, or gene-specific primers.

12. 10. The method of any one of the preceding claims, wherein the second set of sub-portions comprises between 2 and 25,000 sub-portions.

13. 2. A method according to any one of the preceding claims, wherein the second identifier comprises a first and a second sub-identifier.

14. 2. The method of any one of the preceding claims, wherein the second identifier is incorporated into a second strand synthesis reaction.

15. 10. The method of any one of the preceding claims, wherein the cell origin-distinguishing nucleic acid is produced using an amplification-mediated reaction.

16. 10. The method of any one of the preceding claims, wherein the cell origin-distinguishing nucleic acid is produced using a ligation-mediated reaction.

17. 2. The method of any one of the preceding claims, wherein the cell origin-identifiable nucleic acid is produced using a tagmentation-mediated reaction.

18. 2. The method of any one of the preceding claims, wherein the method further comprises lysing the plurality of cell sources of the second set of subportions.

19. 13. The method of any one of the preceding claims, wherein the method further comprises at least one additional pooling / splitting step to produce nucleic acids incorporating at least one further identifier.

20. 2. The method of any one of the preceding claims, wherein the method further comprises sequencing the multiple cell-source-distinguishable nucleic acid collection.

21. 21. The method of claim 20, wherein the sequencing comprises next generation sequencing.

22. 10. The method of any one of the preceding claims, further comprising assigning a cell source to a cell-source identifiable nucleic acid collection based on at least the first and second identifiers of nucleic acids of the collection.

23. 2. The method of any one of the preceding claims, wherein the cell source is an immune cell source.

24. 24. The method of claim 23, wherein the source of immune cells is a T cell or a nucleus thereof.

25. 24. The method of claim 23, wherein the source of immune cells is a B cell or a nucleus thereof.

26. 2. A method according to any one of the preceding claims, wherein the number of sub-portions in the first and second sets is the same.

27. A method according to any preceding claim, wherein the number of sub-portions in the first and second sets is different.

28. 28. The method of claim 27, wherein the number of sub-portions in the second set exceeds the number of sub-portions in the first set.

29. A kit comprising: a plurality of distinct template switch oligonucleotide compositions, each comprising a template switch oligonucleotide comprising a common first identifier, wherein the first identifiers of the template switch oligonucleotides of the different template switch oligonucleotide compositions are different; and a plurality of distinct second identifier nucleic acids.

30. 30. The kit of Claim 29, wherein the multiple distinct template switch oligonucleotide compositions are present in separate containers.

31. 31. The kit of claim 30, wherein the separate containers are wells of a multi-well plate.

32. 32. The kit of any one of claims 29-31, wherein each template switch oligonucleotide of a given template switch oligonucleotide composition further comprises a different unique molecular recognition domain.

33. The kit of any one of claims 29 to 32, wherein the plurality of distinct second identifier nucleic acids are present in separate containers.

34. 34. The kit of claim 33, wherein the separate containers are wells of a multi-well plate.

35. The kit of any one of claims 29 to 34, wherein the second identifier nucleic acid is a primer.

36. The kit of any one of claims 29 to 35, wherein the second identifier nucleic acid is an adaptor.

37. The kit according to any one of claims 29 to 36, further comprising a reverse transcriptase.

38. The kit according to any one of claims 29 to 37, further comprising a polymerase.

39. The kit according to any one of claims 29 to 38, further comprising a ligase.

40. The kit according to any one of claims 29 to 39, further comprising a transposase.

41. The kit according to any one of claims 29 to 40, further comprising a buffer.

42. 1. A method for preparing a multiple cell source distinguishable collection of nucleic acids derived from an initial multiple cell source, the method comprising: (a) providing a first set of cell source sub-portions, each sub-portion including a plurality of cell sources from the initial plurality of cell sources; (b) generating first identifier-tagged nucleic acids in the plurality of cell sources in each subportion of the first set using a template-switching-mediated reaction with a template switch oligonucleotide comprising a first identifier, wherein the first identifiers of the template switch oligonucleotides used in different subportions of the first set are identical within a given subportion but different among the different subportions; (c) pooling the cell sources of the subportions produced in step (b) to generate a first cell source pool comprising a first identifier-tagged nucleic acid; (d) allocating the first cell source pool into a second set of subportions, each subportion including a plurality of cell sources that include the first identifier-tagged nucleic acid; (e) generating, in the plurality of cell sources for each subportion of the second set, second identifier-tagged nucleic acids, wherein second identifiers in different subportions of the second set are identical within a given subportion but different among each of the different subportions; (f) pooling the cell sources of the subportions produced in step (e) to generate a second cell source pool comprising first and second identifier tagged nucleic acids; (g) allocating the second pool of cell sources into a third set of subportions, each set including a plurality of cell sources including the first and second identifier-tagged nucleic acids; (h) producing cell-source-identifiable nucleic acids from the plurality of cell sources in each subportion of the third set of subportions, the nucleic acids comprising a first identifier, a second identifier, and a third identifier, wherein the third identifier of each subportion of the third set is identical within a given subportion but different between different subportions; Thereby, a plurality of cell-source-identifiable nucleic acid collections are prepared from said initial plurality of cell sources, wherein the nucleic acids of each cell-source-identifiable nucleic acid collection comprise a unique combination of first, second and third identifiers that identify the cell source of said nucleic acid.

43. 43. The method of claim 42, wherein step (e) is carried out using a second strand synthesis reaction to add the second identifier.

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