Method to amplify complementary DNA strands

Abstract

A cDNA amplification method is disclosed that can comprehensively amplify mRNA from even extremely small cell samples with high efficiency and low bias. The complementary DNA chain amplification method of the present invention comprises the steps of: capturing target RNA and synthesizing a complementary DNA chain in a liquid or solid system using a reverse transcriptase having terminal transferase activity; adding a base such as CCC to the end of the complementary DNA chain; and performing homopolymer tailing by a TdT reaction in the presence of a chain-stopping nucleotide triphosphate. By further combining a template switching reaction, the complementary DNA chain can be amplified with even higher efficiency and low bias.

Description

Technology Field

[0001] The present invention relates to a method for amplifying a complementary DNA chain that enables high-sensitivity amplification of cDNA from a trace amount of mRNA. Background Technology

[0002] Currently, comprehensive gene expression analysis using micro-samples, such as individual single cells, is being performed. This technology enables single-cell RNA-seq analysis (scRNA-seq analysis) that comprehensively identifies the properties of individual cells in units of thousands to hundreds of thousands, leading to the identification of diversity within cell populations, the identification of new cell subpopulations, and the identification of specific cell populations associated with pathological conditions. It is also serving as a driving force in promoting an understanding of biology at unprecedented resolutions, such as the international project to create a one-cell level atlas in humans, which started in 2016 (Non-patent Literature 1).

[0003] scRNA-seq analysis is particularly useful for elucidating complex immune mechanisms in pathological conditions, and representative trace samples include, for example, tumor-infiltrating T cells contained in biopsy specimens from primary and metastatic sites collected for the diagnosis of cancer patients, or B cells that specifically respond to viruses or vaccines against them. The actual status of cytotoxic T lymphocytes (CTLs), which are the essence of immunotherapy such as anti-PD-1 antibodies, is CD8 + From the perspective of T cells, CD8 cells possessing cancer antigen-specific recognition ability +Attention is focused on methods for measuring the extent to which T cells are induced (Non-patent Literature 2). In addition, regarding the SARS-CoV2 pandemic since 2019 and the development of effective vaccines against them, attention is focused on investigating the properties of B cells with high antibody production capacity that have strong neutralizing activity against the virus, and CD4 / CD8 T cells that react specifically to antigens. One of the measurement methods involves analyzing the sequences of T cell receptors (TCRs) on T cells or B cell receptors (BCRs) on B cells to determine the TCR / BCR repertoire (types of clones and the frequency of existence of each clone) and simultaneously performing scRNA-seq analysis to discuss antigen-specific clone responses and differences in cell properties (scTCR / BCR / RNA-seq). In fact, it has been reported that changes in the TCR repertoire correlate with the therapeutic effect of anti-CD4 antibodies on cancer, and that the types of T cells change following multiple vaccinations against SARS-CoV2 (Non-patent Literature 3, 4). However, for the gene expression of each cell type responsible for such immune responses to be discussed at the same level, it is ideal for the mRNA amounts of the cells to be equal, but the mRNA content of T / B cells is significantly lower compared to epithelial cells, tumor cells, macrophages, etc. For this reason, the importance of developing a method to amplify individual one-cell-derived mRNA from samples with high sensitivity for high-resolution scTCR / BCR / RNA-seq analysis of those cells is increasing.

[0004] Various techniques for cDNA amplification from micro-samples are known. If cDNA amplification methods are classified based on whether mRNA capture and cDNA synthesis are performed in a solid phase, examples of solid-phase systems that capture mRNA in micro-wells or micro-droplets and synthesize cDNA using mRNA capture oligos immobilized on solid beads include Beckton Dickinson’s BD Rhapsody system (Patent Document 3), Seq-well system (Non-Patent Document 12), and Nx1-seq system (Patent Document 5, Non-Patent Document 14) using plates with micro-wells, as well as the Drop-seq method (Non-Patent Document 13) that captures mRNA in micro-droplets and Dolomite Bio’s Nadia system. Examples of liquid-phase systems that synthesize cDNA by capturing mRNA in microwells or microdroplets using free mRNA capturing oligos that are not bound to a solid phase include the 10X Chromium system of 10X Genomics (Patent Document 4), in which a hydrogel bead containing mRNA capturing oligos and a cell are enclosed in a microdroplet, and the hydrogel is melted and the cell is lysed within the microdroplet to capture mRNA with a free mRNA capturing oligo, or Smart-Seq2 (Non-Patent Document 15), in which individual cells or trace amounts of mRNA are dispensed into a 96-well plate and amplified. As an additional example of a liquid-phase analysis technique, combination indexing such as sci-RNA-seq3 can be cited, in which fixed cells are suspended in a solution containing a cell membrane permeability reagent and an mRNA capturing oligo, and mRNA within the fixed cells is captured on the mRNA capturing oligo.

[0005] In addition, if we classify cDNA amplification methods from trace samples based on reaction modes such as second chain synthesis reactions, there is a template switching (TS) method in which, when synthesizing cDNA from mRNA, a primer of tens of base pairs containing a base sequence such as [guanosine]-[guanosine]-[guanosine] is added to the 3' end of the cDNA, which is called a template switching oligo, thereby adding a desired base sequence to the cDNA end and performing second chain synthesis (Patent Document 1, Non-Patent Document 5, Non-Patent Document 6), or a TdT method in which a polynucleotide sequence consisting of the same bases is added to the synthesized cDNA end by terminal deoxynucleotidyl transferase (TdT), and then a primer having a sequence complementary to the polynucleotide at the 3' end and a known adapter sequence at the 5' end is used to perform second chain synthesis (Non-Patent Documents 7-9).

[0006] In addition to the advantage of having less loss of cDNA associated with purification due to the small number of reaction steps, the TS method has the advantage of easily generating full-length cDNA because the TS reaction mainly occurs in mRNA with a 5' cap structure. On the other hand, the efficiency of the TS reaction depends more heavily on the terminal structure of the mRNA, and the efficiency decreases in the order of terminal bases G > A > C > U. While the efficiency is relatively high for full-length mRNA with a cap structure at the 5' end (in the range of about 20%-60%), the efficiency is low for incomplete-length mRNA without a cap structure, immature mRNA, or partially degraded mRNA (in the range of several%-40%), and non-uniform efficiency causes amplification bias (Non-patent Literature 6). Furthermore, there is a drawback that the efficiency is lower than the polynucleotide addition efficiency in the TdT method (92-95%, Non-patent Literature 10).

[0007] On the other hand, the TdT method has better efficiency compared to the TS method, but it has the drawback that it is difficult to control the reaction of TdT, the number of steps is large, and when a single-stranded DNA is used as a template, the efficiency can be significantly reduced if the end forms a higher-order structure and becomes a depressed end (Non-patent Literature 10), so it is more difficult to synthesize long-stranded full-length cDNA, which is easier to form a higher-order structure, than the TS reaction.

[0008] As a technology that solves the difficulty of controlling the TdT reaction in the TdT method, there is the TAS-Seq method previously developed by the inventors of the present invention (Patent Document 2, Non-Patent Document 11). The TAS-Seq method is a solid-phase cDNA amplification method that captures target nucleic acids in a solid phase and synthesizes cDNA. By adding a certain amount of chain-stop nucleotide triphosphate to the TdT reaction system and probabilistically controlling the TdT reaction, the nucleotide homopolymer addition reaction caused by the TdT reaction is controlled to remain within a certain range even under various conditions. Consequently, the TAS-Seq method is a technology that offers excellent detection sensitivity of expressed genes and quantification of cell composition, and can more reliably detect inter-cell communication, which is difficult to detect with conventional technology. Prior art literature

[0009] WO 2015 / 027135 A1WO 2021 / 006353 A1US 2018 / 0258500 A1WO 2015 / 200893 A2WO 2015 / 166768 A1

[0010] Regev A., et al. The Human Cell Atlas. eLife 2017; e27041.Zaretsky JM, Garcia-Diaz A, Shin DS, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med 2016; 375:819-2.Aoki H., et al. TCR Repertoire Analysis Reveals Mobilization of Novel CD8+ T Cell Clones Into the Cancer-Immunity Cycle Following Anti-CD4 Antibody Administration. Front Immunol. 2019 Jan; 9(3185).Aoki H., et al.. T cell responses induced by SARS-CoV-2 mRNA vaccination are associated with clonal replacement bioRxiv. 2022 Aug.Picelli S., et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014 Jan; 9(1):171-81.Wulf MG., et al. Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other. J Biol. Chem. 2019 Nov; 294(48):18220-18231.Matsunaga H, Goto M, Arikawa K, et al. A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.Anal Biochem 2015; 471:9-16.Sasagawa Y., Nikaido I., Hayashi T., Danno H., Uno, K. D., Imai T., Ueda H. R., Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity. Genome Biol 2013 Apr; 14(4)Tang F, Barbacioru C, Wang Y, et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 2009, 6(5):377-382Deng G, Wu R. An improved procedure for utilizing terminal transferase to add homopolymers to the 3' termini of DNA. Nucleic Acid Res 1981, 9(16):4173-4188.Shichino S, Ueha S, Hashimoto S, et al. TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq. Commun Biol. 5:602-602, 2022.Gierahn TM et al. Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat Methods 2017; 14(4):395-398.Macosko EZ et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 2015; 161(5):1202-1214.Hashimoto et al.Scientific Reports 2017 Oct 27; 7(1): 14225. doi:10.1038 / s41598-017-14676-3.Picelli S., et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. Jan 2014; 9(1):171-81. The problem to be solved

[0011] A method to amplify individual one-cell-derived mRNA from various samples with high sensitivity and low bias is an important task in terms of constructing a more sophisticated and precise one-cell atlas, capturing tumor-responsive T cells, and capturing B cells with high vaccine responsiveness. However, as described above, existing TS and TdT methods each have their own unique advantages and disadvantages, and a high-sensitivity cDNA amplification method capable of encompassing all advantages does not yet exist. In the TAS-Seq method developed by the inventors of the present invention, there is a problem in that a certain amount of cDNA is produced in which polynucleotides of the length required to form a stable complementary chain with the primers used for second chain synthesis are not added due to the nature of adding a certain amount of terminator nucleotides. Since the TAS-Seq method is a type of TdT method, it has more steps compared to the TS method, and it is difficult to synthesize full-length long-chain cDNA compared to the TS reaction, just like the general TdT method. In addition, the known TAS-Seq method is a solid-phase cDNA amplification method, and its application to liquid-phase systems is unknown.

[0012] The present invention aims to provide a cDNA amplification method capable of comprehensively amplifying mRNA from even extremely small cell samples with high efficiency and low bias. means of solving the problem

[0013] When the TS method and the TdT method are combined, it is expected that the advantage of the TS method, which is an increase in the quantity of cDNA due to the small number of reaction steps, will be lost. Additionally, because the 3' end of the cDNA is exposed during the template switching reaction of the TS method, the end structure may not be a protruding end desirable for the TdT reaction, and thus the advantage of the TdT method's good reaction efficiency will be lost. Furthermore, in the liquid-phase TS method, in order to avoid a reduction in the product during the purification process, the removal of unreacted (non-targeting) target RNA capturing oligos is not performed after cDNA synthesis (Non-patent Literature 15). That is, in the liquid-phase TS method, if a purification process is performed to remove unreacted target RNA capturing oligos after cDNA synthesis, the cDNA product is reduced, leading to a decrease in the final cDNA amplification efficiency. Meanwhile, according to this technical common sense, if the TdT method is applied directly to the product of the TS method without performing a step to remove unreacted target RNA capture oligos, it is expected that unreacted target RNA capture oligos or template switching oligos, which are present in much larger quantities than the cDNA product, will be preferentially amplified by the TdT method, and the cDNA amplification efficiency will be significantly reduced. Therefore, a method combining the TS method and the TdT method is not seen, nor is there any basis to expect that a significant advantage outweighing the advantage is obtained.

[0014] However, the inventors of the present invention deliberately applied the TdT method to the product of the TS reaction after performing a process to remove unreacted template switching oligos along with unreacted target RNA capturing oligos in the liquid phase, and surprisingly found that the amount of cDNA could be significantly increased compared to the reaction alone, the amount of long-chain cDNA synthesized increased compared to the TdT reaction alone, and the amount of short-chain cDNA synthesized increased compared to the TS reaction alone.

[0015] In addition, the inventors of the present invention have discovered that when the TAS-Seq step (TdT method) is performed without combining the TS reaction by using a reverse transcriptase having terminal transferase activity, the proportion of homopolymer parts (especially poly C) that are excessively short is reduced by adding bases such as CCC to the cDNA ends, so that the TAS-Seq method can be efficiently performed even in a liquid phase system that does not use a solid phase.

[0016] The present invention, completed by reviewing the above examples, is a method for amplifying a complementary DNA chain comprising the steps of: capturing a target RNA and synthesizing a complementary DNA chain in a liquid or solid system using a reverse transcriptase having terminal transferase activity; adding a base such as CCC to the end of the complementary DNA chain; and performing homopolymer tailing by a TdT reaction in the presence of a chain-stopping nucleotide triphosphate. It is a technique capable of amplifying a complementary DNA chain with higher efficiency and lower bias by further combining a TS reaction using a template-switching oligo, and includes the following embodiments.

[0017] [1] Target RNA capture process that captures target RNA by a target RNA capture oligo including a target capture unit;

[0018] A complementary chain synthesis process that synthesizes a complementary DNA chain with a sequence complementary to the captured RNA by performing a reverse transcription reaction using a reverse transcriptase having terminal transferase activity, and adds an addition sequence consisting of any number of bases to the 3' end of at least a portion of the complementary DNA;

[0019] Unreacted oligo removal process for removing target RNA capturing oligos that are not capturing target RNA;

[0020] RNA degradation process in which RNA is degraded by RNA degrading enzymes;

[0021] A homopolymer addition process of performing a reaction with terminal deoxynucleotidyl transferase in the presence of dATP, dTTP, dCTP, or dGTP and a chain-stopping nucleotide triphosphate, and adding a nucleotide homopolymer to the 3' end of the complementary DNA chain;

[0022] A second chain synthesis process for performing second chain synthesis on the complementary DNA chain using a primer for second chain synthesis comprising a primer having a complementary homopolymer portion having a sequence complementary to the nucleotide homopolymer, and producing a DNA double chain composed of a complementary DNA chain and a second chain; and

[0023] A method for amplifying a complementary DNA chain, comprising a nucleic acid amplification process that performs a nucleic acid amplification reaction using the above DNA double chain as a template.

[0024] [2] A target RNA capture oligo includes a first adapter portion on the 5' side of the target capture portion, and a primer for second chain synthesis includes a long primer including a second adapter portion on the 5' side of the complementary homopolymer portion, and in a nucleic acid amplification process, a nucleic acid amplification reaction is performed using a primer targeting the first adapter and a primer targeting the second adapter. [1] The method described.

[0025] [3] A long primer for synthesizing a second chain, comprising a second adapter portion on the 5' side of a complementary homopolymer portion, and a method of [1] described in which a nucleic acid amplification reaction is performed using a primer targeting the second adapter and a primer targeting a desired region of the complementary DNA chain.

[0026] [4] A method of [2] or [3] described in which a long primer comprises a molecular barcode portion consisting of a random sequence between a second adapter portion and a complementary homopolymer portion.

[0027] [5] A method described in any one of [2] to [4] in which the chain length of the complementary homopolymer portion of the long primer is 6 to 15 bases.

[0028] [6] A method described in any one of [1] to [5] in which a target RNA capture oligo is a free oligo that is not bound to a solid carrier, and an unreacted oligo removal process is performed by nucleic acid size fractionation.

[0029] [7] A method of [6] comprising a first adapter portion, a cell identification barcode portion, and a target capture portion, with the target RNA capture oligo extending from the 5' side toward the 3' side.

[0030] [8] A solidified oligo in which a target RNA capture oligo is bound to a solid carrier, and the process of removing unreacted oligos is performed by exonuclease treatment. [1] to [5] A method described in any one of [1] to [5].

[0031] [9] The method described in [8], wherein the above solid carrier is a bead, and the target RNA capture oligo comprises a first adapter portion, a bead identification barcode portion, and a target capture portion extending from the 5' side toward the 3' side.

[0032]

[10] The above-described method, in which the solid carrier is a plate, a target RNA capture oligo is immobilized in a plurality of compartments of the plate, and includes a first adapter portion, a compartment identification barcode portion, and a target capture portion extending from the 5' side toward the 3' side. [8]

[0033]

[11] A method described in any one of [1] to

[10] , wherein the target RNA capture oligo comprises a target poly-A RNA capture oligo comprising a poly-T portion as the target capture portion, and the target RNA comprises a poly-A RNA.

[0034]

[12] In a process for synthesizing a complementary chain, a reverse transcription reaction is performed in the presence of a template switching oligo that includes a sequence capable of hybridizing with the addition sequence at the 3' end and a switching sequence at the 5' side of the sequence, the template switching oligo is hybridized to the complementary DNA chain to which the addition sequence is added, and a sequence complementary to the switching sequence is added to the 3' end of the complementary DNA chain.

[11] Method described.

[0035]

[13] A template switching oligo is a sequence that can be hybridized with the above additional sequence and includes an oligonucleotide having GGG, GUG, or NGG at the 3' end (G and U are bases of ribonucleotide, N is a base of any one ribonucleotide selected from A, U, G and C, and may include one or more nucleotide analogs), and the switching sequence includes a second adapter portion

[12] .

[0036]

[14] A method of

[13] described that includes a molecular barcode portion in which the switching sequence is a random sequence on the 3' side of the second adapter portion.

[0037]

[15] A method described in any one of

[12] to

[14] , wherein the target RNA capturing oligo is a free oligo that is not bound to a solid carrier, and the unreacted oligo removal process comprises removing the target RNA capturing oligo that is not capturing the target RNA and the template switching oligo that is not hybridizing to the complementary DNA chain by nucleic acid size fractionation.

[0038]

[16] A method described in any one of

[11] to

[15] comprising a long primer having a second adapter portion on the 5' side of a complementary homopolymer portion and a short primer having a second adapter portion on the 3' side that does not have a complementary homopolymer portion.

[0039]

[17] A method of

[16] described that includes a molecular barcode portion consisting of a random sequence between a second adapter portion and a complementary homopolymer portion, in which a long primer is included.

[0040]

[18] A method described in any one of

[12] to

[17] in which, in the process of synthesizing complementary chains, a reverse transcription reaction is performed using a reverse transcriptase having additional chain substitution activity in the presence of the template switching oligo, and a second chain synthesis is performed on the complementary DNA chain hybridized by the template switching oligo.

[0041]

[19] A method described in any one of [1] to

[18] in which the chain-stopping nucleotide triphosphate is ddNTP, a derivative of ddNTP, 3'-dNTP, or 3'-deoxy-5-methyluridine-5'-triphosphate.

[0042]

[20] A derivative of ddNTP, a ddNTP in which the 3' position is modified by a non-OH group, as described in

[19] .

[0043]

[21] The method described in

[19] where the chain-stopping nucleotide triphosphate is a ddNTP.

[0044]

[22] A method described in any one of [1] to

[21] for adding poly C by adding dCTP and chain stop CTP in a homopolymer addition process.

[0045]

[23] A method described in any one of [1] to

[22] in which the RNA degradation process and the homopolymer addition process are carried out simultaneously. Effects of the invention

[0046] According to the present invention, by using a reverse transcriptase having terminal transferase activity, mRNA can be comprehensively amplified from an extremely small amount of sample with higher efficiency and lower bias than the conventional TAS-Seq method (Patent Document 2, Non-Patent Document 11), and the TAS-Seq method can be efficiently carried out even in a liquid-phase system that does not use a solid phase. When combined with a TS reaction, the yield of both long-chain cDNA and short-chain cDNA can be significantly increased. In particular, regarding the liquid-phase TS method, according to conventional technical knowledge, it is expected that the cDNA amplification efficiency will decrease whether a purification process to remove unreacted oligos after cDNA synthesis is performed or not performed; however, contrary to this expectation, high efficiency and low bias can be achieved, and the yield of both long-chain cDNA and short-chain cDNA can be significantly increased. According to the method of the present invention, the precision of determining therapeutic effects using, for example, expression analysis or TCR analysis, and the isolation and identification of rare cells (e.g., cancer cells) by scRNA-seq analysis can be significantly improved compared to conventional methods, thereby contributing further to the suppression of medical resource waste. Brief explanation of the drawing

[0047] FIG. 1 is a schematic of the steps of a liquid-phase nonTS / TAS-Seq method using a reverse transcriptase having terminal transferase activity, which is one embodiment of the present invention. FIG. 2 is a schematic of the steps of a solid-state TS / TAS-Seq method using a reverse transcriptase having terminal transferase activity but not chain substitution activity, which is one embodiment of the present invention. FIG. 3 is a schematic of the steps of a solid-state TS / TAS-Seq method using a reverse transcriptase having terminal transferase activity and chain substitution activity, which is one embodiment of the present invention. FIG. 4 is a schematic of the steps of a liquid-phase TS / TAS-Seq method using a reverse transcriptase having terminal transferase activity but not chain substitution activity, which is one embodiment of the present invention. FIG. 5 is a schematic of the steps of a liquid-phase TS / TAS-Seq method using a reverse transcriptase having terminal transferase activity and chain substitution activity, which is one embodiment of the present invention. Figure 6 shows a comparison of the total quantity of cDNA amplification products by the TdT method and the template-switch + TdT method in a solid-state system (BD Rhapsody single-cell analysis system). The total quantity of cDNA is significantly higher for the template-switch + TdT method. Figure 7 shows a comparison of the size distribution of the total cDNA amplification products by the TdT method and the template-switch + TdT method in a solid-state system (BD Rhapsody single-cell analysis system). The template-switch + TdT method has a larger amount of long-chain cDNA. Figure 8 shows a comparison of the quantity and size distribution of total cDNA amplification products by the TdT method and the template-switch + TdT method in a liquid phase system (10X Chromium single-cell analysis system). The template-switch + TdT method has a larger quantity of cDNA and a larger quantity of long-chain cDNA. Figure 9 shows a comparison of the total quantity of cDNA amplification products by the template-switch method and the template-switch + TdT method in the liquid-phase system (Smart-seq2 method). The quantity of cDNA is significantly higher in the template-switch + TdT method. Figure 10 shows a comparison of the size distribution of the total cDNA amplification products by the template-switch method and the template-switch + TdT method in a liquid-phase system (Smart-seq2 method). The template-switch + TdT method has a larger proportion of cDNA with shorter chain lengths within the total cDNA. Figure 11 shows a comparison of the number of detected genes and reads in each cell of 1-cell RNA sequence analysis data by the TdT method, random priming method, and template-switch + TdT method in a solid-state system (BD Rhapsody single-cell analysis system). Under conditions where the distribution of reads per cell is the same, the template-switch + TdT method can detect the most genes. Figure 12 shows a comparison of the number of detected genes and reads in each cell of 1-cell RNA sequence analysis data by the template-switch method and the template-switch + TdT method in a liquid system (10X Chromium single-cell analysis system). Under conditions where the distribution of reads per cell is the same, the template-switch + TdT method can detect more genes. Figure 13 shows the results of one-cell clustering analysis of one-cell RNA sequence analysis data by the template-switch method and the template-switch + TdT method in a liquid system (10X Chromium single-cell analysis system). The template-switch + TdT method has improved cell population separation ability. Figure 14 shows a comparison of the number of detected genes when the number of reads in each cell subset is changed for 1-cell RNA sequence analysis data by the template-switch method and the template-switch + TdT method in a liquid system (10X Chromium single-cell analysis system). In all cell populations, the template-switch + TdT method can detect more genes. Specific details for implementing the invention

[0048] In the present invention, the A, T, G, and C constituting the base sequences of the target RNA capture oligo, template switching oligo, primer, adapter, nucleotide homopolymer, etc. include not only deoxyribonucleotides (A, T, G, C of DNA) constituting DNA, but also ribonucleotides (A, T, G, C of RNA) constituting RNA, and furthermore, the corresponding nucleotide analogs (e.g., bridged nucleic acids such as LNA, ENA, PNA, etc., or modifying bases such as Super T (5-hydroxybutinyl-2'-deoxyuridine), Super G (8-aza-7-deazaguanosine), deoxyinosine, 5-methyl dC, deoxyuridine, 2,6-diaminopurine, 2-aminopurine, 2-amino-dATP, etc.) are also included. That is, the target RNA capture oligo (e.g., a target capture portion, a first adapter portion, a barcode portion, and any one of the other portions), the template switching oligo (a 3' end portion capable of hybridizing with an additional sequence, a second adapter portion, a barcode portion, and any one of the other portions), the nucleotide homopolymer, the primer for second chain synthesis (e.g., a complementary homopolymer portion, a second adapter portion, a barcode portion, and any one of the other portions), and each primer used in the nucleic acid amplification process may contain one or more monomers selected from ribonucleotides and nucleotide analogs (e.g., about 1 to 15, about 1 to 12, or about 1 to several).

[0049] In the present invention, "a few" means a plurality of 2 to 9. 1 to a few may be 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0050] In the present invention, when a primer targeting a certain region X is referred to as such, it means a primer that specifically hybridizes to this region X or its complementary chain. A primer targeting region X includes a primer containing the same sequence as region X or a sequence complementary thereto. Likewise, the term "primer set for region X" refers to a primer that specifically hybridizes to region X or its complementary chain, and includes a primer containing the same sequence as region X or a sequence complementary thereto.

[0051] In the present invention, the terms complementary DNA chain and cDNA refer to a DNA chain consisting of a base sequence complementary to a target RNA, and are not limited to a DNA chain consisting of a sequence complementary to mRNA.

[0052] In the present invention, the words oligonucleotide or oligo refer to a single-chain polynucleotide with a relatively short chain length. The chain length of the oligonucleotide is typically tens of bases to about 200 bases, for example, about 50 bases to 150 bases, but is not limited thereto.

[0053] In the present invention, the term "terminal transferase activity" refers to the activity of terminal deoxynucleotidyl transferase in adding a nucleotide to the 3' end of a nucleotide chain in a template-independent manner. Among the 3' end of a single-strand nucleotide, the 3' protruding end of a double-strand nucleotide, and the smooth end of a double-strand nucleotide, the 3' end becomes the target for nucleotide addition by terminal transferase activity.

[0054] In the present invention, the target RNA includes various RNAs, including poly-A RNA. Poly-A RNA is RNA having a poly-A tail at the 3' end, and the most typical example is mRNA. The cell from which the target RNA originates is not particularly limited, and various cells are subject to use, for example, cells from mouse individuals, various cultured cells, cells from healthy individuals, cells from cancer patients, cells from patients with various diseases, etc. Cells from cancer patients may be cells from cancer patients undergoing cancer immunotherapy. Cells from healthy individuals or patients may be cells collected from peripheral blood or lesion sites (e.g., tumors). When the amplification method of the present invention is used in an analysis method such as transcription analysis, cells may be used as samples, or cell nuclei isolated from various cells may be used as samples.

[0055] The complementary DNA chain amplification method of the present invention comprises a target RNA capture process, a complementary chain synthesis process, an unreacted oligo removal process, an RNA degradation process, a homopolymer addition process, a second chain synthesis process, and a nucleic acid amplification process. The amplification method of the present invention includes an embodiment in which, in the complementary chain synthesis process, a reverse transcription reaction is performed in the presence of a template switching oligo and a template switching (TS) reaction is performed, and an embodiment in which a reverse transcription reaction is performed in the absence of a template switching oligo. Hereinafter, for convenience, the former embodiment is referred to as the TS / TAS-Seq method, and the latter embodiment as the nonTS / TAS-Seq method. As an embodiment of the amplification method of the present invention, the TS / TAS-Seq method is more preferred. The nonTS / TAS-Seq method is preferred when significantly undesirable products (e.g., strand invasion products caused by the template switching oligo, cDNA reverse phase complement products resulting from mispriming of the synthesized cDNA, etc.) are generated by the TS reaction. Both the TS / TAS-Seq method and the nonTS / TAS-Seq method include, in the target RNA capture process, a solid-phase system in which target RNA is captured by a target RNA capturing oligo bound to a solid phase such as beads, and a liquid-phase system in which target RNA is captured by a free target RNA capturing oligo not bound to the solid phase. Figure 1 is a schematic of the liquid-phase nonTS / TAS-Seq method. Figures 2 to 5 are schematics of the TS / TAS-Seq method, where Figures 2 and 3 are the solid-phase system and Figures 4 and 5 are the liquid-phase system.

[0056] Hereinafter, with appropriate reference to the drawings, each process will be explained in the order of the solid-state TS / TAS-Seq method, the liquid-state TS / TAS-Seq method, and the non-TS / TAS-Seq method.

[0057] A. Solid-state TS / TAS-Seq method

[0058] A-1. Target RNA capture process (Step 1 of FIG. 2 and FIG. 3)

[0059] In the target RNA capture process of the solid-state TS / TAS-Seq method, target RNA is captured by a target RNA capture oligo bound to a solid carrier. The target RNA is captured on the solid carrier via the target RNA capture oligo. As described below, the target RNA capture process is carried out in a micro-well or micro-droplet, or in a solution that does not correspond to these.

[0060] The solid carrier may be beads or a plate such as a slide glass. As single-cell analysis systems using a bead solid carrier, examples include Beckton Dickinson’s BD Rhapsody system (Patent Document 3), Seq-well system (Non-Patent Document 12), and Nx1-seq system (Patent Document 5, Non-Patent Document 14) using a plate with micro-wells, as well as the Drop-seq method (Non-Patent Document 13) which captures mRNA in micro-droplets and Dolomite Bio’s Nadia system. In addition, as a system using a plate as a solid carrier, a platform for spatial transcription is also known, in which a tissue sample is permeated onto a plate on which target capture oligos are aligned and immobilized, and mRNA from each cell of the tissue sample is captured by the capture oligos on the plate; for example, there is the Stereo-seq system (Chen et al. Cell 2022 185, 1777-1792. doi:10.1016 / j.cell.2022.04.003.). Solid-state TS / TAS-Seq methods are applicable to any of these known technologies.

[0061] The material of the beads is not particularly limited, and various materials used as nucleic acid capture carriers in nucleic acid analysis kits or solid particle carriers in immunoassay kits can be adopted. Examples of bead materials include organic polymer beads such as resin beads made of polystyrene and polypropylene; semiconductor beads such as quantum dots (semiconductor nanoparticles) made of semiconductor materials such as cadmium selenide (CdSe), zinc sulfide (ZnS), and zinc oxide (ZnO); metal beads such as gold; and polymer beads such as silica beads. Specific examples include beads made of materials such as cellulose, cellulose derivatives, acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, divinylbenzene cross-linked polystyrene (see Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex gel, polystyrene, dextran, rubber, silicone, plastic, nitrocellulose, cellulose, natural sponge, silica gel, glass, metal plastic, cellulose, cross-linked dextran (e.g., Sephadex (trade name)) and agarose gel (Sepharose (trade name)). Either a non-magnetic or magnetic material may be used, but magnetic beads, which are easier to handle, may be preferred.

[0062] The material of the plate is not particularly limited, and various materials used as substrates for microarrays, for example, can be adopted. A representative example of a usable material is glass, but silicon, plastic, etc., can also be used. Solid carriers that serve as plates include various plate-shaped carriers with flat or uneven surfaces, such as microwell plates with aligned microwells, plates used as substrates for microarrays, and flow cells for sequencers. When the solid carrier is a plate, target RNA capture oligos are typically immobilized in multiple compartments on the plate (typically aligned and immobilized in a spot shape).

[0063] As one embodiment of a target RNA capture process in a solid-state system, an embodiment may be provided in which RNA is extracted from individual cells within micro-wells or micro-droplets using a scRNA-seq analysis platform utilizing solid beads, and poly-ARNA or mRNA as target RNA is captured on beads within micro-wells or micro-droplets by contacting them with a bead carrier to which a target RNA capture oligo is bound. In the terms micro-wells and micro-droplets, the word "micro" typically refers to a volume of about 5 to 100 pl. Examples of general methods for scRNA-seq analysis platforms using solid beads are as described above, and include Beckton Dickinson’s BD Rhapsody system (Patent Document 3), Seq-well system (Non-Patent Document 12), and Nx1-seq system (Patent Document 5, Non-Patent Document 14) using plates with micro-wells, the Drop-seq method (Non-Patent Document 13) for capturing mRNA in micro-droplets, and Dolomite Bio’s Nadia system, and these general methods may be preferably used in the present invention.

[0064] As another embodiment of the target RNA capture process in a solid-state system, a tissue sample is permeated onto a plate on which target RNA capture oligos are aligned and immobilized using a spatial transcription platform, and poly-A RNA to mRNA eluted from each cell of the tissue sample is captured by the capture oligos on the plate. An example is the Stereo-seq system (Chen et al. Cell 2022 185, 1777-1792. doi:10.1016 / j.cell.2022.04.003.). This is an embodiment in which the capture of target RNA is performed in a solution whose volume does not conform to the definition of a microscopic value as described above.

[0065] As another embodiment of the target RNA capture process in a solid system, a poly-ARNA sample or mRNA sample is prepared from a cell sample, such as a lesion tissue sample or a cell suspension, and poly-ARNA is captured on a solid carrier by bringing the poly-ARNA sample or mRNA sample into contact with a solid carrier to which a poly-ARNA capture oligo is bound. This is an embodiment in which the capture of target RNA is performed in a solution whose volume does not conform to the definition of a microscopic value as described above. In this embodiment, the preparation of the poly-ARNA sample or mRNA sample may be performed by first extracting total RNA from the cell sample and then extracting the poly-ARNA or mRNA, or the poly-ARNA or mRNA may be extracted directly without undergoing the extraction of total RNA from the cell sample.

[0066] In the TS / TAS-Seq method, the target RNA includes poly-A RNA. It may target only poly-A RNA, or it may also target RNA other than poly-A RNA. That is, in the TS / TAS-Seq method, the target RNA capturing oligos may include target poly-A RNA capturing oligos and may also include target RNA capturing oligos that capture RNA other than poly-A RNA. Furthermore, in addition to the target RNA capturing oligos, it may additionally include target capturing oligos that capture nucleic acid molecules other than RNA. One or more types of target RNA capturing oligos may be immobilized on the solid phase, and target capturing oligos that capture nucleic acid molecules other than RNA may also be immobilized. An example of a target capturing oligo that captures nucleic acid molecules other than RNA is an oligonucleotide that captures labeled oligo DNA in scRNA-seq analysis technology using oligo DNA labeled antibodies, such as BioLegend’s TotalSeq. A technology combining such oligo DNA labeled antibodies with the TdT method is also known (Patent Document 2). In addition, as another example of a target capture oligo that captures nucleic acid molecules other than RNA, an oligonucleotide that captures a DNA fragment recombined by a Tn5 transposase in a one-cell chromatin structure analysis technique using a Tn5 transposase can be cited. In an embodiment in which multiple types of capture oligos are used in combination, if the solid phase is a bead, multiple types of capture oligos may be immobilized on a single bead, or multiple types of capture oligos may be immobilized on different beads. If the solid phase is a plate, multiple types of capture oligos may be immobilized in a single compartment, or multiple types of capture oligos may be immobilized in different compartments.

[0067] Typical examples of target poly-A RNA capture oligonucleotides include an oligonucleotide containing a poly-T portion as a target capture site and capturing a poly-A portion of the target RNA. Examples of target RNA capture oligonucleotides that capture a region other than poly-A include an oligonucleotide containing a random sequence of 6 to 15 bases as a target capture site, or an oligonucleotide containing a sequence complementary to a desired region of a specific RNA sequence (e.g., a normal region of a T cell receptor, a normal region of a B cell receptor, a region near the 5' end of RNA, etc.) as a target capture site.

[0068] A target RNA capture oligo has its 5' end directly or indirectly bound to a solid carrier and has a target capture portion at its 3' end. The oligo may include a first adapter portion on the 5' side of the target capture portion, i.e., on the solid carrier side. The portion indicated as Universal 1 in FIGS. 2 and 3 is the first adapter portion. The first adapter portion becomes a region that sets one side of the primer in the nucleic acid amplification process.

[0069] Additionally, the target RNA capture oligo may include a barcode portion for identifying individual solid carriers or locations on solid carriers between the first adapter portion and the target capture portion. This barcode portion is a bead identification barcode if the solid carrier is a bead, and a compartment identification barcode for identifying which compartment on the plate it is immobilized in if the solid carrier is a plate. That is, the target RNA capture oligo may include the first adapter portion, the bead or compartment identification barcode portion, and the target capture portion extending from the 5' side toward the 3' side. In an embodiment including such a barcode portion, the complementary DNA chain amplified in the nucleic acid amplification process contains a barcode sequence derived from the identification barcode portion. The bead identification barcode portion has the same sequence on the same bead and has a different sequence for each bead. By this, cDNA derived from mRNA captured on the same bead can be distinguished from cDNA derived from mRNA captured on a different bead. In scRNA-seq analysis, cDNA derived from the same cell can be distinguished from cDNA derived from different cells. A solidified poly-A RNA capture oligo containing such a bead identification barcode portion can be prepared by known methods (see, for example, WO 2015 / 166768 A1, etc.). Furthermore, the compartment identification barcode portion has the same sequence within the same compartment (spot) on the plate, but has different sequences for each compartment (spot). By this, for example, when the sample is a tissue specimen, it is possible to determine which mRNA is expressed in which part of the tissue.

[0070] Typically, multiple target RNA capture oligo molecules are immobilized within a single bead or a single compartment. FIGS. 2 and 3 show a solid phase that is a bead, and a target RNA capture oligo that is a target poly-A RNA capture oligo containing a poly-T portion as a target capture portion, and a mode of capturing mRNA as a target poly-A RNA is shown (in the drawings, the poly-T portion of the capture oligo is partially omitted). For convenience, a mode of capturing one mRNA molecule on a single bead carrier is shown, but in reality, multiple mRNA molecules are captured on a single bead carrier. Not only full-length mRNA having a 5' cap structure, but also "non-full-length mRNA" such as incomplete-length mRNA without a cap structure, immature mRNA, or partially degraded mRNA, are captured on the solid phase by the target poly-A RNA capture oligo if they have a poly-A.

[0071] A-2. Complementary chain synthesis process (Step 2 of FIG. 2 and FIG. 3)

[0072] After the target RNA capture process, the solid carrier is washed, and the complementary chain synthesis process is performed with the target RNA captured on the solid carrier. In the complementary chain synthesis process of TS / TAS-Seq, a reverse transcriptase having terminal transferase activity is used, and the reverse transcription reaction and the template switching reaction are performed simultaneously (in a single step) by carrying out the reverse transcription reaction in the presence of a template switching oligo. The timing of adding the template switching oligo to the reaction system is not particularly limited; it may be before, simultaneously with, or after the start of the reverse transcription reaction, or it may be added after a certain period of time has elapsed during the reverse transcription reaction. The reverse transcription reaction and the template switching reaction themselves may be performed according to standard procedures.

[0073] Reverse transcriptases with terminal transferase activity are widely known and various types are commercially available. Representative examples include wild-type MMLV RT, Thermo Fisher Scientific’s SuperScript II, SuperScript IV, Maxima H minus reverse transcriptase, Takara Bio’s SmartScribe reverse transcriptase, and New England Biolabs’ template switching RT.

[0074] By means of a reverse transcription reaction, a DNA chain with a sequence complementary to it (complementary DNA chain, first chain) is synthesized from target RNA captured on a solid carrier. Subsequently, by the terminal transferase activity of the reverse transcriptase, an addition sequence consisting of any number of bases is added to the 3' end of at least some of the synthesized complementary DNA. It is known that in a template-independent base addition reaction to the 3' end by terminal transferase activity, the number of bases added is generally within the range of 1 to 5 bases, and 3 bases are often added, and that when the template RNA is a full-length mRNA having a cap structure at the 5' end, CCC is easily added to the 3' end of the cDNA. A template switching oligo is an oligonucleotide that includes a sequence capable of hybridizing with this addition sequence at its 3' end, and also includes a switching sequence on the 5' side of the said sequence. Therefore, in this process, the template switching oligo hybridizes to the complementary DNA chain to which the additional sequence has been added, and as the complementary DNA chain is extended to the 5' end of the template switching oligo, the complementary chain of the switching sequence is further added to the 3' end of the complementary DNA.

[0075] Preferably, the template switching oligo comprises an oligonucleotide having GGG, GUG, or NGG at the 3' end as a sequence capable of hybridizing with an addition sequence (G and U are ribonucleotide bases, N is a ribonucleotide base selected from A, U, G, and C, and may include one or more nucleotide analogs). FIGS. 2 and 3 show an example in which the hybridizable 3' end sequence is the ribonucleotide base GGG. Two or more template switching oligos with different 3' end sequences may be mixed and used.

[0076] The switching sequence may include a second adapter portion. The part indicated as Universal 2 in the drawing is the second adapter portion. The second adapter portion becomes a region for setting one of the primers in a subsequent nucleic acid amplification process.

[0077] The switching sequence may include a molecular barcode portion consisting of a random sequence on the 3' side of the second adapter portion (between the second adapter portion and the 3' end sequence in the template switching oligo). DNA molecules amplified in a subsequent nucleic acid amplification process will have the same molecular barcode if they originate from the same double-stranded cDNA, and will have different molecular barcodes if they originate from different double-stranded cDNAs. By counting only the DNA with different molecular barcodes, it becomes possible to estimate the number of cDNA molecules at the start or to correct for differences in PCR amplification efficiency between different cDNAs. The chain length of the molecular barcode is not particularly limited, but is typically about 12 to 30 bases. To make the molecular barcode resistant to sequence errors, it may be configured with a specific fixed sequence inserted within the random base sequence. As described below, the long primer used as a primer for second chain synthesis in the second chain synthesis process may also include a similar molecular barcode portion.

[0078] The 5' end or 3' end of the switching sequence (either one or both) may include a chemical modification or a base to prevent polymerization of the template switching oligo, such as an amino modification, a biotin modification, a C3 spacer modification, etc.

[0079] As shown in the first step of Step 2 of FIGS. 2 and 3, when cDNA is synthesized over the entire length of the mRNA on a target RNA capture oligo that has captured full-length mRNA, a CCC is added to the 3' end of the cDNA with high probability due to the terminal transferase activity of the reverse transcriptase. When a template switching oligo with a 3' end sequence of GGG is hybridized to this CCC portion, a complementary chain of the switching sequence is synthesized consecutively to the CCC at the 3' end of the cDNA due to the DNA-dependent DNA polymerase activity of the reverse transcriptase (template-switched cDNA). In the illustrated example, the complementary chain of Universal 2, which is the second adapter, is synthesized, but when a molecular barcode is included between Universal 2 and rGrGrG of the template switching oligo, a molecular barcode complementary chain + Universal 2 complementary chain is synthesized consecutively to the CCC at the 3' end of the cDNA. As a result of the synthesis of the complementary chain of the switching sequence, the end of the mRNA-cDNA hybrid is smoothed, so any number of bases (indicated as NNN in the figure) can be added to the 3' end of the cDNA chain by the terminal transferase activity of reverse transcriptase (in some cases, no addition may be made).

[0080] cDNA is synthesized for the entire length of full-length mRNA, and after CCC is added to the 3' end, cDNA that has not been hybridized by the template switching oligo, or cDNA that has had other bases added to the 3' end that have not been hybridized by the template switching oligo, remains on the solid phase as cDNA that has not undergone the template switching reaction (step 2, 2nd step, non-switched cDNA).

[0081] On the target RNA capture oligo that captures mRNA that is not full length, random number bases (NNN) other than CCC are likely to be added to the ends of the synthesized cDNA (although there are cases where they are not added), so this cDNA also remains on the solid phase as cDNA that has not undergone the template switching reaction (step 3 of step 2, non-switched cDNA).

[0082] Even if full-length mRNA is captured, if cDNA synthesis stops midway, the cDNA ends of the mRNA-cDNA hybrid become indented ends. In this case, since base addition to the cDNA ends does not occur, they do not undergo a template switching reaction and remain in the solid phase (Step 4 of Step 2, unswitched cDNA).

[0083] In the complementary chain synthesis process, it is also possible to use a reverse transcriptase that has additional chain substitution activity in addition to terminal transferase activity. In this case, as shown in Step 2 of FIG. 3, in the hybrid double chain of template-switched cDNA and mRNA, the second chain synthesis proceeds while stripping mRNA from cDNA by the chain substitution activity of the reverse transcriptase.

[0084] Reverse transcriptases having terminal transferase activity and chain substitution activity are also known and various types are commercially available. Representative examples include wild-type MMLV RT, Thermo Fisher Scientific’s SuperScript II, SuperScript IV, Maxima H minus reverse transcriptase, Takara Bio’s SmartScribe reverse transcriptase, and New England Biolabs’ template switching RT.

[0085] A-3. Removal of unreacted oligosaccharides (Step 3 of FIG. 2 and FIG. 3)

[0086] After the complementary chain synthesis process, the solid carrier is washed to proceed to the unreacted oligo removal process. In this process, target RNA capturing oligos (unreacted capturing oligos) that are not capturing target RNA and remain in the solid phase are removed. In the solid phase, template switching oligos (unreacted template switching oligos) that are not hybridized to the complementary DNA chain and remain in the reaction solution are removed by washing the solid phase; therefore, unreacted capturing oligos in the solid phase are degraded and removed by exonuclease treatment. Examples of general exonucleases that specifically degrade single-stranded DNA include exonuclease I and exonuclease T. In the present invention, at least one of such general exonucleases may be used. After exonuclease treatment, the solid carrier is washed to proceed to the next process.

[0087] A-4. RNA degradation process (Step 4 of FIG. 2 and FIG. 3)

[0088] In the RNA degradation process, the mRNA of the mRNA-cDNA hybrid bound to the solid carrier is degraded using an RNA degrading enzyme. If the 3' end sequence of the template switching oligo is a ribonucleotide base, the 3' end portion is also degraded. In this process, general RNA degrading enzymes such as RNase H can be used, and alkaline treatment with NaOH solution, KOH solution, etc., can also be used. After RNA degradation, the solid carrier may be washed and recovered before proceeding to the homopolymer addition process, or the homopolymer addition process may be performed without washing. As described below, the RNA degradation process and the homopolymer addition process may be performed simultaneously.

[0089] A-5. Homopolymer addition process (Step 4 of FIG. 2 and FIG. 3)

[0090] In the homopolymer addition process, a reaction is carried out with terminal deoxynucleotidyl transferase (TdT) in the presence of dATP, dTTP, dCTP, or dGTP and chain-stopping nucleotide triphosphate (chain-stopping NTP), and a nucleotide homopolymer is added to the 3' end of the complementary DNA chain synthesized on the solid phase (Step 4 of FIG. 2 and FIG. 3; the figure shows an example of poly C). For both the template-switched cDNA and the unswitched cDNA, the nucleotide homopolymer is added. As is well known in this field, a chain stop NTP (chain stop ATP, chain stop TTP, chain stop CTP, or chain stop GTP) is a nucleotide modified or altered such that the OH group at the 3' position of the nucleotide cannot form a phosphate ester bond with the 5'-phosphate portion of another nucleotide molecule, and can also be described as a nucleotide triphosphate that does not have an OH group at the 3' position (the atomic group bound to the 3' position does not contain an OH group). As specific examples of general chain-stopping NTPs that can also be used in the present invention, dideoxynucleotide triphosphate (ddNTP) (ddATP, ddCTP, ddGTP, ddTTP, ddUTP), derivatives of ddNTP (typically ddNTPs in which the 3' position is modified by an atomic group not having an OH group, e.g., 3'-azido-ddATP, 3'-azido-ddCTP, 3'-azido-ddGTP, 3'-azido-ddTTP, 3'-azido-ddUTP, 3'-amino-ddATP, 3'-amino-ddCTP, 3'-amino-ddGTP, 3'-amino-ddTTP, etc., ddNTPs in which the 3' position is modified by an azido group or an amino group), 3'-deoxynucleotide triphosphate (3'-dNTP) (3'-dATP, 3'-dCTP, Examples include 3'-dGTP, 3'-dTTP, 3'-dUTP), 3'-deoxy-5-methyluridine-5'-triphosphate, for example, ddNTP, or ddNTP with the 3' position modified by an azido group, particularly ddNTP, but not limited to these.It is preferable that the base chains of the chain stop NTP and the substrate (dNTP) forming the homopolymer used be matched. In order to particularly preferably suppress the minimum homopolymer length required for the second chain synthesis reaction, the required length of the 3' end homopolymer portion of the primer used for the second chain synthesis, and the chain length of by-products derived from pre-primer, it is preferable to perform poly C addition or poly G addition by a combination of chain stop CTP such as ddCTP + dCTP, or chain stop GTP such as ddGTP + dGTP. In the TS / TAS-Seq method using a TS reaction, it is particularly preferable to perform poly C addition by a combination of chain stop CTP + dCTP, in that the cytosine base added to the 3' end of the cDNA chain for full-length mRNA during the TS reaction can also be calculated as a homopolymer portion.

[0091] The amount of chain-stopping NTP added, for example when using ddNTP, should be approximately ddNTP:dNTP = 1:10 to 1:100 in ratio to dNTP (dATP, dTTP, dCTP, or dGTP), and can be used in ratios such as 1:10 to 1:80, 1:10 to 1:60, 1:10 to 1:40, 1:15 to 1:80, 1:15 to 1:60, or 1:15 to 1:40. The amount of other chain-stopping NTPs can also be set based on these ratios. In the case of the conventional TdT method, which performs the TdT reaction without the coexistence of chain-stopping NTPs, it is necessary to strictly control the reaction time according to differences in enzyme activity between lots or differences in the amount of substrates such as primers or cDNA in order to control the chain length of the homopolymer. Meanwhile, in the TAS-Seq method developed by the inventors of the present invention, by performing a homopolymer addition reaction by TdT in the presence of a chain stop NTP, the chain stop NTP is introduced stochastically and the homopolymer elongation reaction is stopped, thereby significantly increasing the allowable amount for differences in reaction time or enzyme activity caused by TdT.

[0092] The homopolymer addition reaction by TdT is generally carried out in the presence of a divalent cation. As for available divalent cations, Zn 2+ , Cu 2+ , Ni 2+ , Co 2+ , Mn 2+ , Mg 2+ Examples include divalent metal cations such as Co 2+ , Mn 2+ or Mg 2+ It is fine, but it is not limited to these people.

[0093] Since the allowable amount for differences in reaction time, etc., caused by TdT is greatly increased by the addition of the chain stop NTP, the RNA degradation reaction and the homopolymer addition reaction can be carried out simultaneously by adding RNase H to the TdT reaction solution. Accordingly, in one embodiment of the present invention, the RNA degradation process and the homopolymer addition process are carried out simultaneously.

[0094] A-6. Second chain synthesis process (Step 5 of FIG. 2 and FIG. 3)

[0095] After the homopolymer addition reaction, the solid carrier is washed to proceed to the second chain synthesis process. The primers for second chain synthesis used in the second chain synthesis process of the TS / TAS-Seq method include a primer (hereinafter referred to as a long primer) that includes a complementary homopolymer portion having a sequence complementary to the nucleotide homopolymer added to the end of the complementary DNA first chain, and also includes a second adapter portion on the 5' side of the complementary homopolymer portion, and a primer (hereinafter referred to as a short primer) that does not include a complementary homopolymer portion and includes a second adapter portion on its 3' side. Since the template-switched cDNA derived from full-length mRNA contains the complementary chain of the second adapter (Universal 2) derived from the template-switching oligo, both the long primer and the short primer can be hybridized. Generally, since the inner primer is easier to elongate, the drawing shows the elongation pattern from the short primer. A long primer hybridizes to the unswitched complementary DNA strand, and the second strand is synthesized by extension. Through the synthesis of the second strand, a DNA double strand consisting of the complementary DNA (first strand) and the second strand is formed on the solid phase. If the template switching oligo contained a molecular barcode, the molecular barcode is inserted into the second strand synthesized in this process with respect to the template-switched cDNA first strand.

[0096] The complementary homopolymer region is located at the 3' end of the long primer. The chain length of the complementary homopolymer region is typically about 6 to 15 bases, for example, about 7 to 13 bases, when the homopolymer tail attached to the first chain of the complementary DNA is poly C or poly G, and typically about 15 to 30 bases, for example, about 18 to 25 bases, when the homopolymer tail is poly A or poly T. Additionally, an anchor sequence of about 1 to 2 bases may be attached to the 3' end of the complementary homopolymer region. By adding an anchor sequence, the probability of the long primer annealing at the initiation site of the homopolymer tail attached to the cDNA by TdT can be increased. For the anchor sequence, if the complementary homopolymer portion is poly G, H or HN (H = A, T or C; N = any base(A, T, C or G)); if it is poly C, D or DN (D = A, T or G; N = any base(A, T, C or G)); if it is poly A, B or BN (B = T, G or C; N = any base(A, T, C or G)); and if it is poly T, V or VN (V = A, G or C; N = any base(A, T, C or G)).

[0097] The second adapter included in the long primer and short primer is the same as the second adapter included in the template switching oligo, and becomes a region for setting one of the primers in the subsequent nucleic acid amplification process.

[0098] The long primer may include a molecular barcode portion consisting of a random sequence, similar to the template switching oligo. In the long primer, the molecular barcode may be included between the second adapter portion and the complementary homopolymer portion. In the amplification product derived from the template-switched cDNA, the molecular barcode included within the switching sequence of the template-switched oligo functions. In the amplification product derived from the unswitched cDNA, the molecular barcode provided in the long primer functions.

[0099] The polymerase used in the second chain synthesis process may be a general polymerase such as Taq polymerase used in conventional PCR, or a polymerase with chain substitution activity such as a polymerase known as a high-accuracy PCR enzyme or Bst DNA polymerase may be used.

[0100] In the second chain synthesis process, if necessary, after thermal denaturation, the annealing and elongation reaction of the primer for second chain synthesis may be performed in only one cycle, or the cycle may be performed by thermal cycling. In addition, after performing one or multiple cycles of the reaction, a subsequent reaction may be performed by a polymerase having chain substitution activity, such as Bst DNA polymerase.

[0101] A-7. Nucleic acid amplification process (Step 6 of FIG. 2 and FIG. 3)

[0102] After the synthesis of the second chain, the solid carrier is washed, or the process proceeds to the nucleic acid amplification step without washing. In the nucleic acid amplification step, the nucleic acid amplification reaction is performed using the DNA double chain synthesized on the solid carrier as a template (Step 6 of FIGS. 2 and FIGS. 3). Since high accuracy is required in this nucleic acid amplification reaction, it is preferable to use a polymerase, which is generally known as a high-accuracy PCR enzyme. Various high-accuracy PCR enzymes are commercially available. In this process, the nucleic acid amplification reaction may be carried out as is by adding the nucleic acid amplification reaction solution without washing the solid carrier.

[0103] In the mode of employing the first adapter and the second adapter, a nucleic acid amplification reaction can be performed using a set of primers set for those adapters. In the case of scRNA-seq analysis, since mRNA expressed in each cell is comprehensively reverse transcribed and amplified, it is common to perform total cDNA amplification using a set of primers targeting the first and second adapters. In this case, the primer set is typically a set of primers consisting of the first adapter sequence and the second adapter sequence, but it may include any additional sequence or modification by biotin on the 5' side.

[0104] Alternatively, instead of a primer targeting the first adapter, a nucleic acid amplification reaction may be performed using a primer targeting a desired region of the complementary DNA strand. In cases where it is sufficient to amplify only a specific region of a complementary DNA molecule synthesized on a solid carrier, such as in the preparation of a TCR variable region library for T cell receptor (TCR) repertoire analysis, a nucleic acid amplification reaction may be performed using a primer targeting the second adapter and a primer targeting a desired region of the complementary DNA strand (e.g., a primer targeting a normal TCR region).

[0105] The nucleic acid amplification reaction itself may be performed using PCR according to standard procedures. Depending on the preference, one or more than two PCR reactions may be performed. For example, a total cDNA byproduct can be obtained by performing several cycles of 1st PCR using a primer set targeting the first and second adapters, and then performing size selection and purification of the byproduct, followed by performing tens of cycles of 2nd PCR using a primer set targeting the same site, or a set of primers targeting the second adapter and a partial region inside the first adapter, and then performing size selection and purification of the byproduct again. In the 2nd PCR, spacers such as biotin or amine may be attached to the 5' ends of both primers, which facilitates the recovery of the DNA fragment to be sequenced.

[0106] B. Liquid-based TS / TAS-Seq method

[0107] B-1. Target RNA capture process (Step 1 of FIG. 4 and FIG. 5)

[0108] In the target RNA capture process of the liquid-phase TS / TAS-Seq method, target RNA is captured by a free target RNA capture oligo that is not bound to a solid carrier. Even in the liquid-phase method, the target RNA capture process can be carried out in micro-wells or micro-droplets, or in a solution that does not correspond to these.

[0109] Liquid-phase analysis techniques are known, for example, the 10X Chromium system of 10X Genomics (Patent Document 4), which encapsulates a cell and a hydrogel bead containing an mRNA capture oligo in a microdroplet, performs melting of the hydrogel and cell lysis within the microdroplet, and captures mRNA with a free mRNA capture oligo; Smart-Seq2 (Non-Patent Document 15), which dispenses individual cells or trace amounts of mRNA into a 96-well plate and performs amplification; and sci-RNA-seq3, which suspends fixed cells in a solution containing a cell membrane permeability reagent and an mRNA capture oligo, and captures mRNA within the fixed cells onto the mRNA capture oligo. The liquid-phase TS / TAS-Seq method is applicable to any of these known transcription analysis techniques.

[0110] As one embodiment of a target RNA capture process in a liquid system, a target RNA capture oligo is encapsulated in a bead made of a material (such as a hydrogel) that can melt under mild conditions such as room temperature, the bead and a cell are encapsulated in a microdroplet or added to a microwell in a 1:1 ratio, and the target RNA, such as mRNA, is captured on the target RNA capture oligo by melting the bead and lysing the cell in the microdroplet or microwell.

[0111] As another embodiment, a target RNA capturing oligo is added to a solution containing cell lysate or RNA extracted from cells, and target RNA, such as mRNA, is captured on the capturing oligo in the solution. This embodiment includes a method of dispensing individual cells or mRNA extracted therefrom into microwells and capturing target RNA, such as mRNA, on the target RNA capturing oligo within the microwells, a method of trapping individual cells in microdroplets and capturing target RNA on the target RNA capturing oligo within the microdroplets, and further, a method of capturing target RNA on the target RNA capturing oligo in a solution that does not correspond to a microvolume.

[0112] As another embodiment, a fixed cell is suspended in a solution containing a cell membrane permeability reagent and a target RNA capture oligo, and a target RNA, such as mRNA, is captured on the target RNA capture oligo within the fixed cell.

[0113] As explained in the solid-phase section, in the liquid-phase TS / TAS-Seq method, the target RNA also includes poly-A RNA. It may target only poly-A RNA, or it may also target RNA other than poly-A RNA. That is, in the TS / TAS-Seq method, the target RNA capture oligo may include a target poly-A RNA capture oligo, and may also include a target RNA capture oligo that captures RNA other than poly-A RNA. In addition to the target RNA capture oligo, it may further include a target capture oligo that captures nucleic acid molecules other than RNA. When capturing target RNA in a micro-well or micro-droplet, multiple types of capture oligos may be included in a single micro-well or micro-droplet, or multiple types of capture oligos may each be included in different micro-wells or micro-droplets.

[0114] Typical examples of target poly-A RNA capture oligos and target RNA capture oligos that capture regions other than poly-A are similar to the solid-state TS / TAS-Seq method. As shown in Step 1 of FIGS. 4 and FIGS. 5, not only full-length mRNA having a 5' cap structure, but also "non-full-length mRNA" such as incomplete-length mRNA without a cap structure, immature mRNA, or partially degraded mRNA, are captured by a target poly-A RNA capture oligo having a poly-T portion as a target capture site if they have poly-A.

[0115] A target RNA capture oligo has a target capture portion at its 3' end. On the 5' side of the target capture portion, a first adapter portion (represented as Universal 1 in FIG. 4 and FIG. 5) that can be used as a primer setting region in a nucleic acid amplification process may be included, and between the first adapter portion and the target capture portion, a cell identification barcode portion for identifying individual cells from which the target RNA originates may be included. This cell identification barcode is a barcode corresponding to the bead identification barcode portion and the compartment identification barcode portion in a solid-state system. A target RNA capture oligo that is encapsulated together with a cell in the same microwell or microdroplet has a cell identification barcode with the same sequence, and the sequence of the cell identification barcode is different for each microwell or microdroplet. By this, cDNA derived from mRNA captured by the capture oligo in the same microwell or microdroplet can be distinguished from cDNA derived from mRNA captured in a different microwell or microdroplet, and furthermore, cDNA derived from the same cell can be distinguished from cDNA derived from a different cell.

[0116] B-2. Complementary chain synthesis process (Step 2 of FIG. 4 and FIG. 5)

[0117] In the liquid phase system, after the target RNA capture process, purification is not performed, and the process proceeds directly to the complementary chain synthesis process. A reverse transcriptase having terminal transferase activity and a template switching oligo are added to the reaction solution after target RNA capture, and the reverse transcription reaction and the TS reaction are carried out in one step.

[0118] The details of this process and the structure of the template switching oligo are identical to those of the solid-state TS / TAS-Seq. The first step of Step 2 in FIGS. 4 and 5 is the template-switched cDNA. cDNA synthesized for the full length of full-length mRNA but not hybridized by the template switching oligo (the second step of Step 2 in FIGS. 4 and 5), cDNA synthesized for the full length of non-full-length mRNA but not hybridized by the template switching oligo (the third step of Step 2), and cDNA whose synthesis was stopped midway (the fourth step of Step 2) remain in the reaction solution as cDNA that did not undergo the template switching reaction.

[0119] When a reverse transcriptase having additional chain substitution activity in addition to terminal transferase activity is used, as shown in Step 2 of FIG. 5, in the hybrid double strand of template-switched cDNA and mRNA, the synthesis of the second strand proceeds while stripping mRNA from the cDNA by the chain substitution activity of the reverse transcriptase. As a difference from the solid-state system, the 3' end of the synthesized second strand and the 5' end of the cDNA strand are smoothed, so a base can be added to the 3' end of the second strand by the terminal transferase activity of the reverse transcriptase (although there are cases where it is not added).

[0120] B-3. ​​Removal of unreacted oligosaccharides (Step 3 of FIG. 4 and FIG. 5)

[0121] In the case of a liquid-phase system, by purifying the reaction solution in which complementary chain synthesis has been completed by nucleic acid size fractionation, unreacted target RNA capture oligos with extremely short chain lengths can be separated and removed from the complementary DNA chain (mRNA-cDNA hybrid). Unreacted template switching oligos can also be removed in a single step simultaneously with unreacted capture oligos. Purification methods by nucleic acid size fractionation are well known and can be carried out according to standard methods, such as purification by a silica membrane column or purification using magnetic beads to which DNA binds. In this process, removal by exonuclease may be combined with nucleic acid size fractionation, thereby further increasing the removal efficiency of unreacted oligos. General exonucleases that specifically degrade single-stranded DNA can be used, and examples include exonuclease I or exonuclease T. By adding exonuclease to the reaction solution after the complementary chain synthesis is completed and performing purification by nucleic acid size fractionation, the exonuclease can also be removed simultaneously.

[0122] B-4. RNA degradation process (Step 4 of FIG. 4 and FIG. 5)

[0123] RNA degrading enzyme is added to the reaction solution after purification by size fractionation to degrade the mRNA of the mRNA-cDNA hybrid and the 3' end sequence of the template switching oligo in the reaction solution (if the sequence is a ribonucleotide base).

[0124] After degrading RNA by adding an RNA degrading enzyme, reagents such as dNTPs, chain stop NTPs, and TdT may be added to the reaction solution to carry out the subsequent homopolymer addition process; however, it is preferable to add the RNA degrading enzyme and these reagents to the reaction solution simultaneously or sequentially to carry out RNA degradation and homopolymer addition simultaneously. When an alkaline solution such as NaOH or KOH is used in the RNA degradation process, the reaction solution is neutralized after the degradation reaction with the alkaline solution, and then the TdT reaction is carried out.

[0125] B-5. Homopolymer addition process (Step 4 of FIG. 4 and FIG. 5)

[0126] A reaction with TdT is carried out in the presence of dATP, dTTP, dCTP, or dGTP and a chain-stopping NTP, and a nucleotide homopolymer is added to the 3' end of the complementary DNA chain (Step 4 of FIG. 4 and FIG. 5; the figure shows an example of poly C). The details of this process, such as preferred examples of chain-stopping NTPs and the ratio of dNTPs to chain-stopping NTPs, are the same as those described below as in other solid-state TS / TAS-Seq processes.

[0127] FIG. 5 is an embodiment in which the reverse transcriptase used in the complementary chain synthesis process has additional chain substitution activity. In this embodiment, since a second chain (the strand represented by the asterisk of Step 4) is synthesized for the template-switched cDNA in the complementary chain synthesis process, a DNA double chain consisting of the first and second cDNA chains exists in the reaction solution at the start of the homopolymer addition process, and both ends of this DNA double chain are smooth ends or 3' protruding ends (see FIG. 5, Step 2, 1st step). Therefore, in the DNA double chain, a homopolymer can be added not only to the 3' end of the template-switched cDNA first chain but also to the 3' end of the second chain.

[0128] B-6. Second chain synthesis process (Step 5 of FIG. 4 and FIG. 5)

[0129] The reaction solution after RNA degradation and homopolymer addition is purified to remove reagents such as RNA degradation enzymes and TdT, and the process proceeds to the second chain synthesis process. The second chain synthesis process of the liquid-phase TS / TAS-Seq is carried out using primers for second chain synthesis that include long primers and short primers, just as in the solid-phase system. The composition of each primer, preferred conditions, and details of this process are the same as those described below as in other solid-phase TS / TAS-Seq systems.

[0130] As a difference from the solid-state system, in the embodiment where a reverse transcriptase having additional chain substitution activity as shown in FIG. 5 is used, the second chain (an asterisk strand) synthesized in the complementary chain synthesis process for the template-switched cDNA may also have a homopolymer at the 3' end, so a reaction in which a long primer hybridizes to the homopolymer of this strand and extends may also occur, as indicated by the dashed line in Step 5. Since the DNA chain produced by this extension reaction does not contain the adapter sequence required for PCR amplification on its 5' end side, the DNA chain is not amplified when a primer targeting the first and second adapters is used in the subsequent nucleic acid amplification process.

[0131] B-7. Nucleic acid amplification process (Step 6 of FIG. 4 and FIG. 5)

[0132] After the second chain synthesis, the reaction solution is purified as needed to remove excess primers and reagents such as polymerase, and the process proceeds to the nucleic acid amplification process. In a liquid-phase system, this process can be carried out in the same way as a solid-phase TS / TAS-Seq.

[0133] C. nonTS / TAS-Seq method (Fig. 1)

[0134] Although Figure 1 shows an example of a liquid-phase system, it can also be carried out in a solid-phase system. In the complementary chain synthesis process, except for performing the reverse transcription reaction in the absence of a template-switching oligo, using a reverse transcriptase enzyme with terminal transferase activity, and making chain substitution activity unnecessary, the process can be carried out in the same manner as the TS / TAS-Seq method. Below, each process of the non-TS / TAS-Seq method will be explained, focusing on the differences from the TS / TAS-Seq method.

[0135] C-1. Target RNA capture process (Step 1 of Fig. 1)

[0136] In the nonTS / TAS-Seq method, the primary target RNA is mRNA that is a poly-A RNA, and it is generally preferable that the target RNA capture oligo contains a target poly-A RNA capture oligo. However, since the TS reaction using CCC addition to cDNA for full-length mRNA is not used in combination, the effects of this method can be obtained even when nonTS / TAS-Seq is performed targeting only non-poly-A RNA. Therefore, in the nonTS / TAS-Seq method, it is not essential that the target RNA capture oligo contains a target poly-A RNA capture oligo.

[0137] C-2. Complementary chain synthesis process (Step 2 of Fig. 1)

[0138] In the complementary chain synthesis process, a reverse transcriptase having terminal transferase activity is used, and a reverse transcription reaction is performed in the absence of a template switching oligo. By this, a complementary DNA chain for mRNA captured by a solidified or free target RNA capturing oligo is synthesized, and any number of bases are added to the 3' end of at least some of the complementary DNA chains.

[0139] C-3. Removal of unreacted oligosaccharides (Step 3 of Fig. 1)

[0140] In the case of a solid system, it can be carried out in the same way as A-3, and in the case of a liquid system, in the same way as B-3.

[0141] C-4. RNA degradation process (Step 4 of Fig. 1)

[0142] In the case of a solid system, it can be carried out in the same way as A-4, and in the case of a liquid system, in the same way as B-4.

[0143] C-5. Homopolymer addition process (Step 4 of Fig. 1)

[0144] In the case of a solid-phase system, it can be carried out in the same manner as A-5, and in the case of a liquid-phase system, in the same manner as B-5. In the nonTS / TAS-Seq method as well, the homopolymer addition process may be carried out after RNA degradation, or RNA degradation and homopolymer addition may be carried out simultaneously.

[0145] C-6. Second chain synthesis process (Step 5 of FIG. 1)

[0146] For the second chain synthesis, a primer containing a long primer is used. A short primer is unnecessary.

[0147] C-7. Nucleic acid amplification process (Step 6 of Fig. 1)

[0148] It can be carried out in the same way as the nucleic acid amplification reaction in the liquid-phase and solid-phase TS / TAS-Seq methods.

[0149] Complementary DNA strands amplified by the method of the present invention, including nonTS / TAS-Seq and TS / TAS-Seq, may be subjected to processing such as fragmentation, end repair, A-tailing, and the addition of sequence adapters in order to be provided for sequencing by a next-generation sequencer. Through these processing, a library of cDNA processed into sequence constructs is obtained. Ultrasound or enzymes may be used as means for fragmentation. Generally used reagents may be used to prepare sequence constructs; representative enzymatic methods include the NEBNext UltraII FS kit from New England Biolabs or the KAPA HyperPlus kit from KAPA Biosystems, and ultrasonic fragmentation devices such as Covaris or Bioruptor may be used, but other equivalent products may also be used. Regarding sequence adapters, it is preferable to use an adapter suitable for the next-generation sequencer being used.

[0150] Sequencing of the library is generally performed using a sequencer referred to as a next-generation sequencer. Specific examples of next-generation sequencers that can be preferably used include Illumina’s Novaseq 6000 system, Novaseq X system, Hiseq system, Nextseq system, Nextseq2000 system, Nextseq3000 system, and MiSeq system; MGI’s DNBseq T7 system, DNBseq G400 system, and DNBseq G40 system; Thermo Fisher Scientific’s Ion S5 / Ion S5 XL system, etc.

[0151] Examples

[0152] The present invention will be explained in more detail below based on examples. However, the present invention is not limited to the following examples.

[0153] 1. Comparison of the TdT method (TAS-Seq method) and the cDNA amplification method of the present invention in a scRNA-seq analysis system using a solid carrier (Figs. 6, 7)

[0154] A single-cell transcriptome library trapped on solid beads prepared by the BD Rhapsody system (Beckton Dickinson) was amplified by the TdT method (TAS-Seq method, Patent Document 2 and Non-Patent Document 11) and the cDNA amplification method according to the present invention (template-switch + TdT method, or template-switch + TAS-Seq method), respectively. The sequences of the primers used are shown in Table 1 below.

[0155] Human peripheral blood monocytes (PBMCs) were obtained from LONZA after being adjusted and cryopreserved. The frozen human PBMCs were rapidly thawed in a 37°C water bath, and 9 ml of pre-warmed RPMI 1640 / 5% FBS was added to the suspension, mixed by electrolysis, centrifuged, and the supernatant removed. The mixture was then washed again with RPMI 1640 / 5% FBS. The cell count was measured using a flow cytometer, and cell beads were loaded into a microwell cartridge at an appropriate density according to the Rhapsody specifications. The cells were lysed, and one cell-derived mRNA was trapped on each bead (a poly-A RNA capture oligo containing a Rhapsody universal adapter) (target RNA capture process). In addition, the Rhapsody universal adapter solidified on the bead is DNA having the following structure, and CLS1 (cell label section 1) to CLS3 each employ 96 types of known unique sequences consisting of 9 bases, totaling 288 types, and constitute approximately 900,000 bead identification barcode parts in total. In sequence number 1 of the sequence table, CLS1 to CLS3 are represented as NNNNNNNNN.

[0156] <Rhapsody 유니버설 어댑터의 구조>

[0157] ACACGACGCTCTTCCGATCT-[none or A or GT or TCA]-(CLS1)-GTGA-(CLS2)-GACA-(CLS3)-NNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 1)

[0158] Four mRNA-trapped beads were each divided into two (a total of eight), and reverse transcription to cDNA amplification was performed on four by the TAS-Seq method and on the remaining four by the cDNA amplification method of the present invention.

[0159] In the TAS-Seq method, the above-mentioned poly A RNA capture beads were subjected to a reverse transcription reaction as recommended by the manufacturer (using a reverse transcriptase with complementary chain synthesis process, terminal transferase activity, and chain substitution activity), and subsequently, unreacted poly A RNA capture oligos on the beads were removed by exonuclease I treatment (unreacted oligo removal process). Using dCTP and ddCTP as the terminator, mRNA degradation by RNaseH and a homopolymer addition reaction by TdT were carried out simultaneously for 20 minutes. After washing the beads, a second chain synthesis reaction was performed using a primer having a guanine homopolymer at the 3' end of the universal 2 adapter sequence and a high-precision DNA polymerase. The total cDNA was amplified for 9 cycles using PCR with primers for the universal 1 and universal 2 sequences (universal oligo-long, 5'BDWTAv2). After size selection using AmPure XP beads, the amount was further amplified by 5 cycles of PCR. The final amount of cDNA was measured by a Qubit fluorometer or a nanodrop spectrophotometer (Thermo Fisher Scientific), and the size distribution of the cDNA was measured by a MultiNA system (Shimadzu Seisakusho).

[0160] The cDNA amplification method of the present invention involves performing a reverse transcription reaction at 42°C for 30 minutes using the above-mentioned poly A RNA capture beads with a reagent provided by the manufacturer, and then adding a template switching oligo (5'BDWTAv2-TSO) and MgCl2, followed by additional reverse transcription and template switching reactions at 42°C for 30 minutes (complementary chain synthesis process). Afterward, unreacted poly A RNA capture oligos on the beads are removed by treatment with exonuclease I (unreacted oligo removal process). Using dCTP and ddCTP as a terminator, mRNA degradation by RNaseH and a homopolymer addition reaction by TdT are carried out simultaneously for 20 minutes. After washing the beads, a second chain synthesis reaction is carried out using a primer having a guanine homopolymer at the 3' end of a universal 2 adapter sequence and a high-precision DNA polymerase. For the reaction mixture, the total cDNA was amplified for 9 cycles by PCR using primers for universal 1 and universal 2 sequences (universal oligo-long, 5'BDWTAv2). After size selection using AmPure XP beads (Beckman Coulter), it was further amplified by 5 cycles of PCR. The final amount of cDNA was measured by a Qubit fluorometer or a nanodrop spectrophotometer (Thermo Fisher Scientific), and the size distribution of the cDNA was measured by a MultiNA system (Shimadzu Seisakusho).

[0161] Figure 6 shows the results of a comparison of cDNA quantities. In the cDNA amplification method of the present invention, which performs both TS / TdT reactions, significantly more cDNA was obtained compared to the conventional TdT method (TAS-Seq method) which performs only the TdT reaction.

[0162] Figure 7 shows the results of a comparison of cDNA size distributions. Compared to the conventional TdT method (TAS-Seq method) which performs only the TdT reaction, the cDNA amplification method of the present invention, which performs both the TS / TdT reaction, shows a higher peak at 1000-2000 base pairs (bp), suggesting that more long full-length cDNA is synthesized.

[0163]

[0164] 2. Comparison of the TdT method and the cDNA amplification method of the present invention in a liquid-phase scRNA-seq analysis system (Fig. 8)

[0165] Human PBMC specimens were acquired in the same manner as described in 1 above. After counting the cells, the cells were loaded into a microfluidic system at an appropriate density according to the specifications of Chromium using 10X Genomics’ Chromium v3.1 reagent, and each cell and hydrogel bead were encapsulated in a microdroplet. Additionally, when loading cells into the Chromium, a cDNA synthesis reagent containing cell lysis reagents and reverse transcription reagents (using a reverse transcriptase with terminal transferase activity and chain substitution activity) was loaded simultaneously; however, for comparison, specimens containing a template switching oligo (10X Chromium-TSO) and specimens without it were prepared. After lysing the cells, one cell-derived mRNA was trapped in each poly-A RNA capture oligo released from the hydrogel (10X Chromium adapter) (target RNA capture process), and a reverse transcription reaction, or a reverse transcription reaction and a TS reaction, were performed directly within the microdroplet at 37°C or 53°C for 1 hour (complementary chain synthesis process). After the completion of the complementary chain synthesis process, the emulsion constituting the microdroplet was broken down according to the method recommended by 10X, and unreacted poly-A RNA capture oligos and template switching oligos were removed by purifying the cDNA by nucleic acid size fractionation (unreacted oligo removal process). In addition, the 10X Chromium Adapter is a DNA having the structure as follows, and CB is a known identification barcode consisting of 16 bases, with approximately 900,000 variations. In Sequence No. 5 of the sequence table, CB is represented as NNNNNNNNNNNNNNNN.

[0166] Structure of the 10X Chromium Adapter

[0167] CTACACGACGCTCTTCCGATCT- (CB) NNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTVN (SEQ ID NO: 5)

[0168] Based on the obtained cDNA solution, dCTP and ddCTP as terminators were used, and mRNA degradation by RNaseH and homopolymer addition by TdT were carried out simultaneously for 20 minutes. After purifying the TdT reaction solution, a second chain synthesis reaction was performed using a primer having a guanine homopolymer at the 3' end of the Universal 2 adapter sequence and a high-precision DNA polymerase. The total cDNA was amplified for 9 cycles using PCR with primers for Universal 1 and Universal 2 sequences (universal oligo-long, 5'BDWTAv2). After size selection using AmPure XP beads (Beckman Coulter), the obtained cDNA was further amplified by 5 cycles of PCR. The final amount of cDNA was measured using a nanodrop spectrophotometer, and the size distribution of the cDNA was measured using a MultiNA system.

[0169] Figure 8 shows the results of a comparison of the cDNA quantity and size distribution. Compared to the conventional TdT method which performs only the TdT reaction, the cDNA amplification method of the present invention, which performs both the TS / TdT reaction, yields more cDNA, and the peak at 1000-2000 base pairs (bp) is higher, suggesting that more long full-length cDNA is synthesized.

[0170]

[0171] 3. Comparison of the TS method and the cDNA amplification method of the present invention in a liquid-phase cDNA amplification system (Figs. 9, 10)

[0172] NIH3T3 cells were cultured, and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). 10 ng of total RNA (6 samples) was used, a poly-A RNA capture oligo (BioEcoP-dT25-adapter) was added (targeted RNA capture process), and reverse transcription and TS reactions were performed at 42°C for 1 hour using the Smart-seq2 method (Picelli S., et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014 Jan; 9(1):171-81.) (complementary chain synthesis process, using a reverse transcriptase with terminal transferase activity and chain substitution activity). After the completion of the complementary chain synthesis process, cDNA was purified by nucleic acid size fractionation using Pronex beads (Promega) (unreacted oligo removal process). For the third sample, the total cDNA was amplified by 11 cycles of PCR using 5'BDWTAv2 and 3'WTA primers and purified by AmPure XP. For the remaining three samples, mRNA degradation by RNaseH and homopolymer addition by TdT were carried out simultaneously for 20 minutes using dCTP and ddCTP as the terminator. After purifying the TdT reaction solution, a second chain synthesis reaction was performed using a primer having a guanine homopolymer at the 3' end of the Universal 2 adapter sequence and a high-precision DNA polymerase. For the reaction solution, the total cDNA was amplified by 11 cycles of PCR using primers for Universal 1 and Universal 2 sequences (5'BDWTAv2 and 3'WTA primers) and purified by AmPure XP. The final amount of cDNA was measured by a Qubit Fluorometer, the relative content of Actb and Rps3 genes in the cDNA was measured by quantitative real-time PCR (qPCR), and the size distribution of the cDNA was measured by a MultiNA system.

[0173] Figure 9 shows the results of a comparison of cDNA quantities. Compared to amplification using only the TS method (Smart-seq2 method), the cDNA amplification method of the present invention, which performs both the TS / TdT reaction, shows that the total cDNA quantity is significantly increased by about 3 times and the quantities of Rps3 and Actb genes are increased, suggesting that the cDNA synthesis efficiency is greatly increased.

[0174] Figure 10 shows the results of a comparison of representative cDNA distributions. It was confirmed that in the cDNA amplification method of the present invention, which performs both TS / TdT reactions compared to amplification using only the TS method (Smart-seq2 method), the proportion of small cDNAs of size 300-700 bp in the total is increased, suggesting that nucleic acids to which TS reactions are difficult to apply, such as cDNA where reverse transcription stopped midway through mRNA, immature mRNA, or partially degraded mRNA, may be captured in the cDNA amplification method of the present invention.

[0175]

[0176] 4. Comparison of the number of detected genes in the TdT method, random priming method, and the cDNA amplification method of the present invention in scRNA-seq analysis using a solid carrier (Fig. 11)

[0177] Spleens were extracted from 8-week-old mice. The spleens were gently crushed on a 70 µm strainer placed on a 6-well plate filled with 3 ml of ACK buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), and the cell suspension was recovered. 7 ml of DMEM / 5% FBS was added to the suspension, mixed by electrophoresis, centrifuged, and the supernatant removed. The mixture was then washed again with DMEM / 5% FBS. Using the acquired cells, cDNA amplification was performed using the TdT method (TAS-Seq method), the cDNA amplification method of the present invention, and random priming with the BD Rhapsody kit. Reverse transcriptases possessing terminal transferase activity and chain substitution activity were used. After constructing a sequence library from the obtained cDNA, sequencing was performed using an Illumina Novaseq 6000. The primer sequences used for constructing the sequence library are shown in the table. This is shown in Figure 4. The obtained sequence data was mapped to a reference sequence (GRCm38-101), and gene expression data for each cell was obtained. Additionally, for comparison, data acquired from mouse splenocytes using 10X Chromium v3.1 (TS method, accession ID: GSE192930) was downloaded from a public database. For the obtained data, the sequence data was downsampled to ensure uniform median read counts per cell, mapped again to the reference sequence (GRCm38-101), and gene expression data for each cell was obtained.

[0178]

[0179] Figure 11 shows the results of a comparison between the number of detected genes and the number of reads. It was found that more genes were detected in the data (TAS-Seq2) obtained by the cDNA amplification method of the present invention compared to other methods. From this, the usefulness of the amplification method of the present invention in solid-state single-cell transcription analysis was demonstrated.

[0180] 5. Comparison of the number of detected genes between the TS method and the cDNA amplification method of the present invention in scRNA-seq analysis using a liquid system (Figs. 12–14)

[0181] Human PBMC specimens (3 specimens) were acquired in the same manner as described in 1 above. After counting the cells, the cells were loaded into a microfluidic system at an appropriate density according to the Chromium specifications using 10X Genomics Chromium v3.1 reagent, and each cell and hydrogel bead were encapsulated in a microdroplet. Two libraries were prepared for each specimen. After lysing the cells, one cell-derived mRNA from each specimen was trapped in a poly-A RNA capture oligo (10X Chromium adapter) released from the hydrogel (target RNA capture process), and reverse transcription and TS reactions were performed directly within the microdroplet at 53°C for 1 hour (complementary chain synthesis process, using a reverse transcriptase with terminal transferase activity and chain substitution activity). After the completion of the complementary chain synthesis process, the emulsion constituting the microdroplet was broken down according to the method recommended by 10X, and unreacted poly-A RNA capture oligos and template switching oligos were removed by purifying the cDNA by nucleic acid size fractionation (unreacted oligo removal process). Based on the obtained cDNA solution, one of the libraries derived from each of the three samples was amplified as is, with the total cDNA for 9 cycles using PCR with primers for universal 1 and universal 2 sequences (universal oligo-long, 5'BDWTAv2). After size selection using AmPure XP beads (Beckman Coulter), the obtained cDNA was further amplified by 5 cycles of PCR. For the other library derived from each of the remaining three samples, mRNA degradation by RNaseH and homopolymer addition reaction by TdT were carried out simultaneously for 20 minutes using dCTP and ddCTP as the terminator. After purifying the TdT reaction solution, the second chain synthesis reaction was carried out using a primer having a guanine homopolymer at the 3' end of a universal 2 adapter sequence and a high-precision DNA polymerase.For the reaction mixture, the total cDNA was amplified for 9 cycles by PCR using primers for universal 1 and universal 2 sequences (universal oligo-long, 5'BDWTAv2). After size selection using AmPure XP beads (Beckman Coulter), the obtained cDNA was further amplified by 5 cycles of PCR.

[0182] After constructing a sequence library from the obtained cDNA, sequencing was performed using an Illumina Novaseq 6000. The primer sequences used to construct the sequence library are shown in Table 5. The obtained sequence data was mapped to a human reference sequence using 10X Cell Ranger software to obtain gene expression data for each cell. For the obtained data, the sequence data was downsampled to ensure a uniform median read count per cell, mapped again to the human reference sequence, and gene expression data for each cell was obtained.

[0183]

[0184] Figure 12 shows the results of a comparison of the number of detected genes and reads. In the data obtained by the cDNA amplification method of the present invention (10XTAS-Seq2), compared with the data of the TS method alone (10X), it was found that more genes were detected in each of the three samples of the same origin.

[0185] The results of cell clustering are shown in Fig. 13. In the data obtained by the cDNA amplification method of the present invention (10X+TAS-Seq2), compared with the data of the TS method alone (10X), it was found that the separation of various cell populations with different properties in the UMAP space was improved.

[0186] Figure 14 shows a comparison of the number of detected genes and reads in various cell populations based on the results of cell clustering. In all detected cell populations, it was found that the number of detected genes per cell increased in the data obtained by the cDNA amplification method of the present invention (10X+TAS-Seq2) compared to the data obtained by the TS method alone (10X). Since one of the major applications of single-cell transcription analysis is the separation and identification of cell populations and the elucidation of their properties, the usefulness of the liquid-phase single-cell transcription analysis of the present invention in the separation and identification of cell populations and the elucidation of their properties has been demonstrated.

[0187] In addition, according to the present invention, when the analysis cost is the same, the amount of information per cell can be increased by 1.5 to 2 times compared to the conventional method. On the other hand, when obtaining the same amount of information per cell as the conventional method, the required sequence analysis cost can be reduced to 1 / 2 to 1 / 3 times (Fig. 14). In particular, in current scRNA-seq analysis, the number of cells analyzed has increased significantly from tens of thousands to hundreds of thousands, and cell throughput has increased greatly, but the amount of sequence analysis required per cell has not changed; therefore, it is expected that the sequence analysis cost will become a bottleneck in scRNA-seq analysis in the future. The present invention can reduce the sequence analysis cost, which is expected to account for most of the scRNA-seq analysis costs in the near future, by 1 / 2 to 1 / 3 times, thereby increasing cost-effectiveness and contributing significantly to industrial application.

Claims

Claim 1 A target RNA capture process for capturing target RNA using a target RNA capture oligo comprising a target capture portion, which is a free oligoine not bound to a solid carrier; a complement chain synthesis process for synthesizing a complementary DNA chain with a sequence complementary to the captured RNA by performing a reverse transcription reaction using a reverse transcriptase having terminal transferase activity, and adding an addition sequence consisting of any number of bases to the 3' end of at least a portion of the complementary DNA; an unreacted oligo removal process for removing target RNA capture oligos not capturing target RNA by nucleic acid size fractionation; an RNA degradation process for degrading RNA by an RNA degradase; a homopolymer addition process for adding a nucleotide homopolymer to the 3' end of the complementary DNA chain by performing a reaction with a terminal deoxynucleotidyl transferase in the presence of dATP, dTTP, dCTP, or dGTP and a chain-stopping nucleotide triphosphate; and a primer comprising a complementary homopolymer portion with a sequence complementary to the nucleotide homopolymer. A method for amplifying a complementary DNA chain, comprising: a second chain synthesis process for performing second chain synthesis on the complementary DNA chain using a primer for second chain synthesis and producing a DNA double chain composed of the complementary DNA chain and the second chain; and a nucleic acid amplification process for performing a nucleic acid amplification reaction using the DNA double chain as a template. Claim 2 A method according to claim 1, wherein a target RNA capture oligo comprises a first adapter portion on the 5' side of the target capture portion, and a primer for second chain synthesis comprises a long primer comprising a second adapter portion on the 5' side of the complementary homopolymer portion, and wherein, in a nucleic acid amplification process, a nucleic acid amplification reaction is performed using a primer targeting the first adapter and a primer targeting the second adapter. Claim 3 A method according to claim 1, wherein the primer for second chain synthesis comprises a long primer including a second adapter portion on the 5' side of a complementary homopolymer portion, and in a nucleic acid amplification process, a nucleic acid amplification reaction is performed using a primer targeting the second adapter and a primer targeting a desired region of the complementary DNA chain. Claim 4 A method according to claim 2 or 3, wherein the long primer comprises a molecular barcode portion consisting of a random sequence between the second adapter portion and the complementary homopolymer portion. Claim 5 A method according to claim 2 or 3, wherein the chain length of the complementary homopolymer portion of the long primer is 6 to 15 bases. Claim 6 A method according to claim 1, wherein a target RNA capture oligo comprises a first adapter portion, a cell identification barcode portion, and a target capture portion extending from the 5' side toward the 3' side. Claim 7 A method according to claim 1, wherein the target RNA capture oligo comprises a target poly-A RNA capture oligo comprising a poly-T portion as a target capture portion, and the target RNA comprises a poly-A RNA. Claim 8 A target RNA capture process for capturing a target RNA using a target RNA capture oligo comprising a target capture portion; a complementary chain synthesis process for synthesizing a complementary DNA chain with a sequence complementary to the captured RNA by performing a reverse transcription reaction using a reverse transcriptase having terminal transferase activity, and adding an addition sequence consisting of any number of bases to the 3' end of at least a portion of the complementary DNA; an unreacted oligo removal process for removing target RNA capture oligos that are not capturing the target RNA; an RNA degradation process for degrading RNA using an RNA degrading enzyme; a homopolymer addition process for adding a nucleotide homopolymer to the 3' end of the complementary DNA chain by performing a reaction with a terminal deoxynucleotidyl transferase in the presence of dATP, dTTP, dCTP, or dGTP and a chain suspension nucleotide triphosphate; and a second chain synthesis for the complementary DNA chain using a primer for second chain synthesis comprising a primer having a complementary homopolymer portion with a sequence complementary to the nucleotide homopolymer. A method for amplifying a complementary DNA strand, comprising: a second strand synthesis process for producing a DNA double strand composed of a complementary DNA strand and a second strand; and a nucleic acid amplification process for performing a nucleic acid amplification reaction using the DNA double strand as a template, wherein the target RNA capturing oligo comprises a target poly-A RNA capturing oligo comprising a poly-T portion as a target capturing portion, and the target RNA comprises a poly-A RNA, wherein in the complementary strand synthesis process, a reverse transcription reaction is performed in the coexistence of a template switching oligo comprising a sequence capable of hybridizing with the addition sequence at the 3' end and a switching sequence on the 5' side of the sequence, wherein the template switching oligo is hybridized to the complementary DNA strand to which the addition sequence is added, and a sequence complementary to the switching sequence is further added to the 3' end of the complementary DNA strand. Claim 9 A method according to claim 8, wherein the template switching oligo comprises an oligonucleotide having GGG, GUG, or NGG at the 3' end as a sequence capable of hybridizing with the addition sequence (G and U are bases of a ribonucleotide, N is a base of any one ribonucleotide selected from A, U, G, and C, and may include one or more nucleotide analogs), and the switching sequence comprises a second adapter portion. Claim 10 A method according to claim 9, wherein the switching sequence comprises a molecular barcode portion consisting of a random sequence on the 3' side of the second adapter portion. Claim 11 A method according to claim 8, wherein the target RNA capturing oligo is a free oligo that is not bound to a solid carrier, and the unreacted oligo removal process comprises removing the target RNA capturing oligo that is not capturing the target RNA and the template switching oligo that is not hybridized to the complementary DNA chain by nucleic acid size fractionation. Claim 12 A method according to claim 8, wherein the target RNA capturing oligo is a solidified oligo bound to a solid carrier, and the process of removing unreacted oligos is performed by exonuclease treatment. Claim 13 A method according to claim 12, wherein the solid carrier is a bead, and the target RNA capturing oligo comprises a first adapter portion, a bead identification barcode portion, and a target capturing portion extending from the 5' side toward the 3' side. Claim 14 A method according to claim 12, wherein the solid carrier is a plate, a target RNA capture oligo is immobilized in a plurality of compartments of the plate, and comprises a first adapter portion, a compartment identification barcode portion, and a target capture portion extending from the 5' side toward the 3' side. Claim 15 A method according to claim 8, wherein the primer for second chain synthesis comprises a long primer comprising a second adapter portion on the 5' side of a complementary homopolymer portion, and a short primer comprising a second adapter portion on the 3' side that does not include a complementary homopolymer portion. Claim 16 A method according to claim 15, wherein the long primer comprises a molecular barcode portion consisting of a random sequence between the second adapter portion and the complementary homopolymer portion. Claim 17 A method according to claim 8, wherein, in a complementary chain synthesis process, a reverse transcription reaction is performed using a reverse transcriptase having additional chain substitution activity in the presence of the template switching oligo, and a second chain synthesis is performed on the complementary DNA chain hybridized by the template switching oligo. Claim 18 Method of claim 1 or 8, wherein the chain-stopped nucleotide triphosphate is a ddNTP, a derivative of a ddNTP, a 3'-dNTP, or a 3'-deoxy-5-methyluridine-5'-triphosphate. Claim 19 In claim 18, the method wherein the derivative of the ddNTP is a ddNTP in which the 3' position is modified by an atomic group that does not have an OH group. Claim 20 In claim 18, the method wherein the chain-stopped nucleotide triphosphate is a ddNTP. Claim 21 A method according to claim 1 or 8, wherein, in a homopolymer addition process, poly C addition is performed by adding dCTP and chain-stopped CTP. Claim 22 A method according to claim 1 or 8, wherein the RNA degradation process and the homopolymer addition process are carried out simultaneously. Claim 23 delete

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