Method for preparing NGS library enabling simultaneous analysis of t cell receptor (TCR) or b cell receptor (BCR) repertoire and gene expression

Abstract

To provide methods for preparing NGS library to enable simultaneous analysis of T cell receptor (TCR) or B cell receptor (BCR) repertoire and gene expression.SOLUTION: The disclosed method comprises the steps of: (1) capturing mRNA eluted from cell using an RT probe immobilized on a solid phase surface; (2) generating 1st cDNA using the captured mRNA as a template; (3) removing excess RT probe not contributed to the 1st cDNA synthesis; (4) conducting PCR amplification using the 1st cDNA as a template; (5) forming circular DNA on the PCR amplified product using a ligase; (6) conducting PCR amplification using the circular DNA as a template; (7) separating a portion of the PCR amplified product obtained in (4) for gene expression analysis and amplifying at least partial region of the subject genes by PCR.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for simultaneously preparing an NGS library for T cell receptor (TCR) or B cell receptor (BCR) repertoire analysis and an NGS library for gene expression analysis, and a method for simultaneously analyzing the diversity and gene expression of T cell receptor (TCR) or B cell receptor (BCR) repertoire. [Background technology]

[0002] Repertoire analysis is a method for investigating the diversity of the variable domains (VDJ) of the gene TRATRB, which encodes the T cell receptor (TCR) present on the cell surface, and the gene encoding the B cell receptor (BCR), with the aim of analyzing the functions of T lymphocytes and B lymphocytes, which play an important role in immune responses. TCR repertoire analysis is also used to detect minimal residual disease (MRD) in cancer patients. Conventional methods usually involve the use of multiple cells (10 6 One method involves synthesizing cDNA from RNA extracted from multiple cells (e.g., 10 cells) by reverse transcription, preparing a library by multiplex PCR amplification using multiple primers (FW primers) corresponding to the variable region of the TCR gene and RV primers corresponding to the C region sequence, and then analyzing the library with a next-generation sequencer (NGS) (Non-Patent Document 1). 6 One method involves synthesizing cDNA from RNA extracted from a single cell (e.g., a single cell) by reverse transcription using a Template Switching Oligo (TSO), introducing a Template Switching (TS) sequence (known sequence) into the 5' end of the TCR gene (mRNA), and preparing a library by PCR amplification using a set of FW primers corresponding to this TS sequence and RV primers corresponding to the C region sequence, followed by NGS analysis (Non-Patent Document 2). These methods involve bulk analysis using multiple cells, and are useful for detecting MRD and examining clonality.

[0003] On the other hand, TCRα / β (or γ / δ) pair information obtained through single-cell TCR repertoire analysis is useful for T cell therapy and vaccine development. Furthermore, expression information on multiple disease-related genes is useful for identifying cellular states. Recently, a single-cell analysis method capable of simultaneously obtaining TCR repertoire pair information and gene expression information has been reported (Non-Patent Document 2). Single-cell analysis involves analyzing mRNA eluted from a single cell. The method described in Figure 3A of Non-Patent Document 2 isolates cells by encapsulating each cell and bead individually in an oil droplet (emulsion). cDNA can be obtained from mRNA derived from individual cells by reverse transcription using a liquid-phase consensus sequence-added oligo(dT) and a bead-immobilized TSO probe. Specifically, cDNA is synthesized with an amplification sequence, cell identification barcode, molecular identification barcode (UMI), and TS sequence inserted into the 5' end of the mRNA contained in each cell. The emulsion is then broken, and the cDNA is recovered and PCR-amplified using a primer set corresponding to the TS sequence and the common sequence (added to oligo(dT)). This amplified product is used to prepare libraries for repertoire and gene expression, respectively. Specifically, the repertoire library is prepared by PCR amplification using a primer set corresponding to the TS sequence and the C region sequence. The prepared library is then analyzed using a next-generation sequencer (NGS). The method described in Figure 3B of Non-Patent Document 2 involves sorting and fractionating single cells and solid-phase carriers (magnetic beads) into each well of a microplate. Probes consisting of a common sequence, cell identification barcode, UMI, and oligo(dT) sequence are immobilized on the surface of the magnetic beads. mRNA from a single cell is captured on the magnetic bead surface, and cDNA is synthesized directly on the surface. After the magnetic beads are collected from each well into a tube, cDNA is synthesized by reverse transcription using TSO (liquid phase) to which a poly(A) sequence has been added. Therefore, a TS sequence and a polyA sequence are added to the 3' end of the synthesized cDNA (5' end of the mRNA).Next, after hybridization with the oligo(dT) sequence of the probe immobilized on magnetic beads, complementary strand synthesis using a reagent containing DNA polymerase adds a cell identification barcode, UMI, and common sequence to the 5' end of the mRNA. This adds the same common sequence to both ends of the cDNA, which is then amplified by PCR using a primer set corresponding to this common sequence and the C region sequence to prepare a repertoire library. cDNA synthesized from other expressed genes remaining on the bead surface is used as a template, and PCR amplification is performed with a primer set corresponding to the common sequence and gene-specific sequence to prepare a gene expression library. These libraries are then used for next-generation sequencing (NGS) analysis. In both of these methods, the sequences required for single-cell analysis (cell identification barcode and UMI) are introduced into the 5' end of the mRNA, allowing for the preparation of short libraries of less than 1,000 bases to enable NGS analysis of the repertoire variable region.

[0004] A gene expression analysis method (Patent Document 1) has been disclosed in which sequences required for single-cell analysis (cell identification barcodes and UMIs) are introduced into the 5' end of cDNA. This method involves capturing and lysing many single cells in individual micro-reaction chambers within a device, and then performing the processes up to cDNA amplification or nucleic acid amplification on the same device. In this method, multiple micro-reaction chambers are filled with multiple beads immobilized with RT probes consisting of amplification sequences, UMIs, cell identification barcodes, and oligo(dT). Single cells are captured, isolated, and lysed in these micro-reaction chambers. mRNA eluted from the cells is captured by RT probes immobilized on beads or the surface of a porous material. Generally, the total amount of DNA that can bind to beads is proportional to the total surface area of ​​the beads (surface area per bead x number of beads) (estimated from Table 2 in Non-Patent Documents 3 and 4 and other information). Therefore, the bead surface area (total of multiple microbeads) in this method is two orders of magnitude larger than other techniques (methods A and B in Figure 3 of Non-Patent Document 1), and therefore the amount of RT probe required to capture mRNA is two orders of magnitude larger. This allows for highly efficient capture of mRNA. This method analyzes gene expression by performing NGS analysis on a library obtained by PCR amplification of cDNA synthesized by reverse transcription using a primer set containing gene-specific sequences (multiple types) and amplification sequences. This method has a high efficiency of converting mRNA to cDNA, enabling highly sensitive gene expression analysis of a single cell. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US10646869B [Non-patent literature]

[0006] [Non-Patent Document 1] R. Amoriello and C. Ballerini, EBioMedicine 61 (2020) 103021 [Non-patent document 2] Hiroyasu Aoki et al., Frontiers in Immunology 13, Article 807696 (2022) [Non-patent document 3] Invitrogen Catalog Nos. 65001, 65002 (Internet site: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / dynabeads_myone_savC1_man.pdf) [Non-patent document 4] Invitrogen Catalog Nos. 11205D, 11206D, and 60210 (available on the Internet at https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / MAN0014017_Dynabeads_M280_Streptavidin_UG.pdf) Summary of the Invention [Problem to be solved by the invention]

[0007] Along with the research and development of diagnostic and therapeutic methods for cancer and infectious diseases, repertoire analysis, which examines the diversity of the variable regions of T cell receptors (TCR) or B cell receptors (BCR) present on the cell surface, has attracted attention. In particular, with the spread of NGS, high-throughput analysis has become possible, and single-cell analysis has attracted attention as a means of simultaneously obtaining repertoire information and gene expression analysis information.

[0008] For example, as shown in Figure 1 (1), the TCR gene (mRNA) 104 has a variable region 101 (VDJ, approximately 300 to 400 bases) at the 5' end and a long C region 102 (e.g., human TRA: 978 bases, human TRB: 760 bases, human TRD: 720 bases, human TRG: 2730 bases) at the 3' end. The BCR gene also has a variable region (VDJ, approximately 300 to 500 bases) at the 5' end and a long C region at the 3' end (examples of heavy chains: human IGHG1: 2619 bases, human IGHG2: 2594 bases, human IGHG3: 2751 bases, human IGHG4: 2597 bases, human IGHA1: 1310 bases, human IGHA2: 1320 bases, human IGHM: 1686 bases, human IGHD: 1678 bases, human IGHE: 1485 bases; examples of light chains: human IGKC: 523 bases, human IGLC1: 460 bases, human IGLC2: 462 bases, human IGLC3: 462 bases, human IGLC7: 441 bases). As shown in Figure 1(2), reverse transcription using a solid-phase support 106 immobilized with an RT probe 113 consisting of an amplification sequence 112, a cell identification barcode 111, a UMI (molecular identification barcode) 110, and oligo(dT) 109, and a solution-phase TSO 105, allows the synthesis of cDNA 108, which contains a TS sequence 107 at the 5' end of the variable region of the mRNA and a UMI 110, cell identification barcode 111, and amplification sequence 112 at the 3' end of the C region. To perform NGS analysis of the variable region required for repertoire analysis, the cell identification barcode required for single-cell analysis, or the UMI for molecular identification, a region containing all of these must be amplified by PCR. Therefore, as shown in Figure 1(3), conventional PCR amplification using known sequences at both ends (TS sequence, amplification sequence) as FW / RV primer sets 114, 115 yields an amplified product exceeding 1,000 bases in size. Furthermore, to prepare a library for TCR analysis for NGS, NGS consensus sequence 1 116 and consensus sequence 2 117 (88 bases each) must be added to both ends, as shown in Figure 1 (4), and the final DNA size increases to approximately 1,200–1,600 bases (118).

[0009] TCR repertoire analysis at the single-cell level requires sequence information for the TCR variable region (the 3' end of the cDNA) and the cell identification barcode / UMI sequence (the 5' end of the cDNA). However, libraries significantly longer than 1000 bases are difficult to analyze using NGS. Generally, NGS is a short-read instrument, and the maximum sequence length that can be determined is 600 bases. To successfully perform TCR repertoire analysis at the single-cell level using NGS, a challenge is the need to prepare a library containing both (1) the TCR variable region and (2) the cell identification barcode and UMI within a short library of only a few hundred bases. This is because, with commonly used NGS (Illumina), the efficiency of the bridge PCR process performed within the NGS instrument decreases significantly as library sizes increase (especially for libraries longer than 1000 bases), making it difficult to obtain analytical data.

[0010] To solve this problem, methods A and B in Figure 3 of Non-Patent Document 2 employ a method for introducing (2) a cell identification barcode + UMI into the 5' end of mRNA (3' end of cDNA), enabling the preparation of repertoire libraries and gene expression libraries for NGS analysis. However, both methods have the problem of sample loss due to a decrease in reaction efficiency in the steps prior to PCR amplification. To achieve highly sensitive single-cell analysis, a method must be developed for preparing NGS libraries by processing many mRNA molecules derived from a single cell with high efficiency in each step without loss.

[0011] Specifically, in the method shown in FIGURE 3A of Non-Patent Document 2, droplet technology is used to encapsulate a single cell, solid support (gel beads), and reverse transcription reaction solution (enzyme, substrate, oligo(dT), etc.) in approximately 1 nanoliter of GEM (Gel Bead in Emulsion), and cDNA is synthesized from mRNA derived from a single cell. In this process, a probe (68 bases in total) consisting of the common sequence (29 bases), cell identification barcode (16 bases), UMI (10 bases), and TSO sequence (13 bases) is immobilized on the surface of gel beads to introduce the common sequence (29 bases), cell identification barcode (16 bases), UMI (10 bases), and TSO sequence (13 bases) into the 5' end of the mRNA, i.e., the 3' end of the synthesized cDNA. The amount of probe that can be immobilized on the surface of a solid support is limited by the length and surface area of ​​the probe (oligonucleotide). According to Non-Patent Documents 3 and 4, the amount of probe that can be immobilized on the surface of a solid support is limited by the length and surface area of ​​the probe (oligonucleotide). 2 Fixed amount of oligonucleotides (less than 30 bases) per molecule up to 2.4x10 4 The total surface area of ​​the gel beads is estimated to be 1.13 x 10 4 μm 2 (Calculated from the bead diameter of approximately 30 μm) and the length is more than twice as long (68 bases), the total amount of TSO-containing probes immobilized on the gel bead surface and used in the reaction is 2.7 × 10 8 This value is calculated based on the total amount of TSO in a reverse transcription reaction solution (calculated at 1 nL, the same as for GEM) that uses a typical liquid-phase TSO (10 to 5 μM): 3 x 10 9 ~6x10 9 This is an order of magnitude lower than that of the molecule. Therefore, the step of introducing the TS sequence in the method of FIGURE 3A in Non-Patent Document 2 has a problem of sample loss because the reaction efficiency is lower than that of conventional methods.

[0012] In the method shown in Figure 3B of Non-Patent Document 2, single cells and solid supports are sorted and fractionated into each well of a microplate. An RT probe consisting of an amplification sequence, a cell identification barcode, a UMI, and an oligo(dT) sequence is immobilized on the surface of the solid support. The poly(A) tail at the 3' end of mRNA derived from a single cell hybridizes with the oligo(dT) and is captured on the solid support surface. After magnetic beads are collected from each well into a tube, cDNA synthesis is performed by reverse transcription using TSO (liquid phase) with a poly(A) sequence added. This introduces a TS sequence and a poly(A) sequence into the 5' end of the mRNA, i.e., the 3' end of the synthesized cDNA. The poly(A) sequence present at the 3' end of this cDNA hybridizes with the oligo(dT) sequence of the RT probe immobilized on the solid support, followed by complementary strand synthesis using a reagent containing DNA polymerase, which further adds the cell identification barcode, UMI, and consensus sequence to the 3' end of the cDNA. The cDNA synthesized from the TRA or TRB gene is thought to be about 1,500 bases long, including the sequences added to both ends during the reverse transcription reaction. It is thought that this long cDNA molecule (with the polyA sequence added to the 3' end) would bend and not hybridize efficiently with the oligo(dT) on the solid support to which it is immobilized. Furthermore, similar to the method shown in Figure 3A of Non-Patent Document 2, the total amount of RT probe used in the reaction was estimated to be 4.91 x 10 8 Less than a molecule (bead diameter: approximately 40 μm) Total surface area of ​​beads estimated based on: 2.05 x 10 4 μm 2 and oligonucleotide lengths of 60 bases or more), the reaction efficiency is likely to decrease further, leading to the issue of sample loss. [Means for solving the problem]

[0013] The new library preparation method proposed herein can solve the above-mentioned problem: a method for preparing a short NGS library of approximately several hundred bases containing (1) the variable region of TCR and (2) a cell identification barcode (UMI) inserted into the 5' end of cDNA from minute amounts of mRNA (molecules) derived from single cells, with high efficiency and no sample loss. We have found that libraries for repertoire and gene expression can be prepared with high efficiency and ease. Furthermore, we have found that combining this new library preparation method with a single-cell analysis device such as that described in Patent Document 1 enables even more efficient and simple single-cell analysis. We have also found that this library preparation method can be used for bulk analysis, achieving high efficiency and simplicity.

[0014] That is, the present invention encompasses the following: In one embodiment, there is provided a method for simultaneously preparing a library for repertoire analysis of T cell receptors (TCRs) or B cell receptors (BCRs) and a library for gene expression analysis, the method comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) separating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, and amplifying at least a portion of the target gene by PCR using a primer set utilizing the amplification sequence and a sequence specific to the target gene; The present invention provides a method comprising:

[0015] In another aspect, a method for simultaneous analysis of T cell receptor (TCR) or B cell receptor (BCR) repertoire analysis and gene expression analysis, comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) analyzing the sequence amplified by PCR in (6); (8) a step of isolating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, and amplifying at least a portion of the target gene by PCR using a primer set utilizing the amplification sequence and a sequence specific to the target gene; (9) analyzing the sequence amplified by PCR in (8); The present invention provides a method comprising:

[0016] In yet another aspect, there is provided a method for preparing a library for repertoire analysis of a T cell receptor (TCR) or a B cell receptor (BCR), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; The present invention provides a method comprising:

[0017] In yet another embodiment, there is provided a method for analyzing a repertoire of a T cell receptor (TCR) or a B cell receptor (BCR), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) analyzing the sequence amplified by PCR in (6); The present invention provides a method comprising: [Effects of the Invention]

[0018] The method of the present invention allows for the easy preparation of libraries for T cell receptor (TCR) or B cell receptor (BCR) repertoires and gene expression analyses without sample loss. By subjecting the prepared libraries to NGS analysis and integrating the data from both, TCR (or BCR) repertoire analysis and gene expression analysis at the single-cell level become possible. Furthermore, using libraries prepared by the method of the present invention can simplify and enhance sensitivity not only in single-cell analysis but also in bulk analysis using multiple cells. Therefore, the present invention is useful for, for example, MRD detection methods, cell function analysis, disease diagnosis, drug discovery, and other applications where high sensitivity is important. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the structure of the TCR (BCR) gene (mRNA) and the amplification reaction process by a conventional method. [Figure 2] FIG. 1 is a diagram showing a process for preparing a library for a repertoire according to one embodiment of the present invention. [Figure 3] FIG. 1 is a flowchart of a first embodiment. [Figure 4] 1 is a graph showing the self-ligation efficiency using a commercially available ligase. [Figure 5] 1A is a photograph showing the results of electrophoresis of a TCR repertoire library prepared using one embodiment of the present invention, and FIG. 1B is a graph showing the mapping rate to TCR genes in NGS analysis data. [Figure 6] 1 is a graph showing NGS analysis data of a gene expression library prepared using one embodiment of the present invention. [Figure 7] FIG. 10 is a flowchart of a second embodiment. [Figure 8] FIG. 1 is a diagram illustrating an example of the configuration of a device (Patent Document 1) used in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to a method for preparing a library of T cell receptor (TCR) or B cell receptor (BCR) repertoires, and a method for analyzing T cell receptor (TCR) or B cell receptor (BCR) repertoires. TCR or BCR repertoire analysis is a method for analyzing the diversity of the variable regions (VDJ) located on the 5' side of genes (TRA, TRB) encoding TCRs present on the cell surface and / or genes encoding B cell receptors (BCRs). For example, the diversity of TCRs can be measured by a method using a 10 18 This diversity exceeds 100%. By comparing analytical data before and after disease treatment, for example, it is possible to obtain information on changes in immune cells over time. In the method of the present invention, a TCR or BCR repertoire library can be prepared and analyzed at the single-cell level or by bulk analysis. Here, "single-cell level" refers to preparing a library that reflects the combination of TRA genes and TRB genes in a single cell, or the combination of IgA / IgD / IgE / IgG / IgM genes in BCR, or the combination of IgA / IgD / IgE / IgG / IgM genes and IgL / IgK genes, and analyzing the sequences of the combinations of the above genes present in a single cell.

[0021] The present invention also relates to a method for simultaneously preparing an NGS library for T cell receptor (TCR) or B cell receptor (BCR) repertoire analysis and an NGS library for gene expression analysis, and a method for simultaneously analyzing the diversity and gene expression of T cell receptor (TCR) or B cell receptor (BCR) repertoires. Gene expression analysis refers to quantitative analysis of the expression level of a target gene in a cell, preferably quantitative analysis of the presence or absence of expression or the expression level of multiple genes. In the context of the present invention, "simultaneous" refers to preparing a library for TCR or BCR repertoire analysis and a library for gene expression analysis using mRNA captured from the same cell sample, and then performing TCR or BCR repertoire analysis and gene expression analysis, but these analyses do not necessarily have to be performed simultaneously. For example, a library for repertoire analysis may be prepared and analyzed, followed by a library for gene expression analysis, or a library for gene expression analysis may be prepared and then a library for repertoire analysis, and both libraries may be analyzed in a single NGS run.

[0022] The method of the present invention is outlined below. After capturing the TCR or BCR gene (mRNA) with an RT probe immobilized on a solid surface and containing an amplification sequence (a cell identification barcode, in the case of a single cell), a molecular identification barcode (UMI), and oligo(dT), cDNA (first cDNA) is synthesized by reverse transcription using a template switching oligo (TSO). Any RT probe remaining on the solid surface that did not contribute to cDNA synthesis is removed. The cDNA is amplified by PCR using a 5'-phosphorylated primer set corresponding to known sequences (TS sequence and amplification sequence) present at both ends. A portion of this amplified product is self-ligated with ligase to form circular DNA, bringing the (cell identification barcode and) UMI and the TCR variable region into close proximity. The circularized DNA is then PCR-amplified using a FW / RV primer set designed based on the C region to generate short amplification products (several hundred bases) suitable for next-generation sequencing (NGS), containing both (1) the TCR variable region and (2) the cell identification barcode and UMI. This allows for easy, sample-free library preparation for repertoire analysis without sample loss. An NGS library for gene expression analysis is then prepared from the amplification products PCR-amplified using the 5'-phosphorylated primer set described above. By separating and integrating the NGS data by the CT barcode, cell identification barcode, and UMI, simultaneous analysis data (preferably at the single-cell level) can be obtained for repertoire and gene expression.

[0023] In one aspect, the present invention provides a method for simultaneously preparing a library for repertoire analysis of T cell receptors (TCRs) or B cell receptors (BCRs) and a library for gene expression analysis, the method comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) separating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, and amplifying at least a portion of the target gene by PCR using a primer set utilizing the amplification sequence and a sequence specific to the target gene; The present invention provides a method comprising:

[0024] In another aspect, the present invention provides a method for simultaneous analysis of repertoire analysis and gene expression analysis of a T cell receptor (TCR) or a B cell receptor (BCR), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) analyzing the sequence amplified by PCR in (6); (8) a step of isolating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, and amplifying at least a portion of the target gene by PCR using a primer set utilizing the amplification sequence and a sequence specific to the target gene; (9) analyzing the sequence amplified by PCR in (8); The present invention provides a method comprising:

[0025] In yet another aspect, there is provided a method for preparing a library for repertoire analysis of a T cell receptor (TCR) or a B cell receptor (BCR), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; The present invention provides a method comprising:

[0026] In yet another embodiment, there is provided a method for analyzing a repertoire of a T cell receptor (TCR) or a B cell receptor (BCR), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) analyzing the sequence amplified by PCR in (6); The present invention provides a method comprising:

[0027] In one embodiment, the barcode sequence includes a cell identification barcode and a molecular identification barcode (UMI), and the cell is a single cell. This allows library preparation and analysis of TCR or BCR repertoire at the single cell level. In another embodiment, the barcode sequence includes a molecular identification barcode (UMI), and the cell is a plurality of cells. In a further embodiment, the barcode sequence may include a sample identification barcode. For the cell identification barcode, a cell identification barcode having a known sequence that differs for each cell is used. For example, when a random sequence of 5 bases is used, 4 5 It is possible to identify 1024 cells. In other words, in one operation, it is possible to analyze 1024 single cells while identifying the mRNA derived from each cell. Furthermore, for the molecular identification barcode, a molecular identification barcode with a different random sequence is used for each RT probe molecule (mRNA molecule or mRNA-derived DNA molecule). When a molecular identification barcode (for example, 7 bases) is introduced into the probe, 4 7 =1.6×10 5Because the barcodes can identify molecules, it is possible to identify the molecule origin of amplification products that originate from the same cell and have the same gene sequence based on the sequence data of the amplification products obtained by next-generation sequencing (NGS). In other words, the molecular identification barcodes can be used to correct amplification bias, allowing highly accurate quantitative data to be obtained. The cell identification barcodes and molecular identification barcodes are described in detail in, for example, WO2014 / 141386.

[0028] The solid phase is preferably made using a material with a large surface area to increase the efficiency of mRNA capture, and for example, it is preferable to use one or more beads, a porous structure, a mesh structure, etc. When beads are used as the solid phase carrier, they can be made from resin materials (such as polystyrene), oxides (such as glass), metals (such as iron), Sepharose, or combinations thereof. For ease of operation, it is preferable to use magnetic beads (paramagnetic beads). The solid phase carrier is preferably one with a diameter of 10 nm to 10 μm, for example, beads with a diameter of 10 nm to 10 μm.

[0029] An RT probe containing an amplification sequence, a barcode sequence, and an oligo(dT) sequence is immobilized on the surface of the solid phase. Such an RT probe can be synthesized by conventional oligonucleotide synthesis methods and can be immobilized on the solid phase by any method known in the art. The degree of polymerization of the oligo(dT) may be sufficient to hybridize with the poly(A) sequence of the mRNA and capture the mRNA on the solid phase to which the oligo(dT) is immobilized. For example, it can be approximately 10 to 20 bases. By incorporating the amplification sequence into the RT probe, this sequence can be used as a common primer in the PCR amplification process.

[0030] mRNA eluted from cells is captured by an RT probe immobilized on a solid phase. In this specification, "mRNA capture" refers to the extraction of mRNA molecules contained within cells and their separation from other cellular components. Specifically, mRNA is eluted from cells using a cell lysis solution known in the art. For example, cells can be lysed using proteolytic enzymes, chaotropic salts such as guanidine thiocyanate and guanidine hydrochloride, surfactants such as Tween and SDS, or commercially available cell lysis reagents (e.g., lysis solution), and the nucleic acids contained therein, i.e., mRNA, can be eluted. If necessary, the status of cell lysis can be monitored using an observation device. The eluted mRNA is captured by the RT probe via binding to the oligo(dT) sequence of the RT probe.

[0031] Next, the captured mRNA is reverse-transcribed to synthesize a first cDNA having a sequence complementary to the mRNA sequence or a portion thereof. This first cDNA synthesis, i.e., complementary strand synthesis, can be performed by a method known in the art using a reverse transcriptase with template switching function and a template switching oligo (TSO) to synthesize cDNA, and a known sequence (TS) is added to the 3' end of the resulting first cDNA. After the synthesis reaction, the mRNA is degraded and removed, for example, using RNase. As a result, a cDNA library composed of first cDNAs corresponding to the mRNA is produced on the solid phase.

[0032] The device, microreaction chambers, and solid phase are washed, as needed, with washing solutions to remove unwanted components and reagents.

[0033] Excess RT probes immobilized on the solid surface that did not contribute to the synthesis of the first cDNA are removed. For example, excess probes can be removed by digesting them with Exonuclease I.

[0034] Using the generated first cDNA as a template, PCR amplification is performed using a primer set that utilizes an amplification sequence present at the 5' end of the first cDNA and a known sequence (TS) at the 3' end. By performing the amplification reaction in this manner, sample loss can be reduced. Primer design and PCR amplification reactions can be performed routinely by those skilled in the art. In one embodiment, in the PCR amplification step (4), a primer set whose 5' end is phosphorylated is used. This adds a phosphate group to the end of the resulting amplification product, enabling efficient circularization by ligase, as described below.

[0035] Ligase is used to form circular DNA from the amplified products obtained by the PCR amplification step. The ligase used is not particularly limited as long as it can ligate the blunt ends of the amplified products. For example, commercially available ligases listed in Table 1 below can be used.

[0036] Next, using the circular DNA as a template, a region containing the barcode sequence and the variable region (VDJ) of the TCR or BCR is amplified by PCR using a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of the TCR or BCR. In one embodiment, the primer set uses a first primer set for TRA (or TRG) for TCR repertoire analysis and a second primer set for TRB (or TRD) for TCR repertoire analysis. In another embodiment, the primer set uses five types of first primer sets for IgA / IgD / IgE / IgG / IgM for BCR repertoire analysis and two types of second primer sets for IgL / IgK for BCR-light chain analysis. Such primer sets can be designed appropriately by those skilled in the art depending on the target of analysis.

[0037] In one embodiment, a primer set is used that is designed to be located near the 5' end and near the 3' end of the C region sequence so that the region to be PCR-amplified is less than 1000 bases. This ensures that the resulting amplification product has a length suitable for next-generation sequencing (NGS) analysis. Such a primer set can be appropriately designed by those skilled in the art based on the sequence of the C region.

[0038] As a result, a repertoire library of T cell receptors (TCRs) or B cell receptors (BCRs) can be prepared.

[0039] The method of the present invention further includes the step of separating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis and preparing a next-generation sequencer (NGS) library for gene expression analysis. Specifically, at least a portion of the target gene (e.g., the 3' region of mRNA) is amplified by PCR using a primer set designed based on an amplification sequence and a sequence specific to the target gene. The target gene preferably includes multiple genes, and libraries can be prepared in parallel for multiple genes whose gene expression analysis is desired. This allows a TCR or BCR repertoire library and a library for gene expression analysis to be prepared in parallel.

[0040] In the method of analyzing a repertoire and gene expression of a T cell receptor (TCR) or a B cell receptor (BCR) according to the present invention, a library for the repertoire and a library for gene expression analysis are prepared as described above, and then PCR-amplified sequences are analyzed. The analysis is not particularly limited as long as it is next-generation sequencer (NGS) analysis.

[0041] The method of the present invention further includes the steps of isolating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, preparing a next-generation sequencer (NGS) library for gene expression analysis, and analyzing the sequence of the library. Specifically, at least a portion of the gene of interest (e.g., the 3'-terminal region) is amplified by PCR using a primer set designed based on the amplification sequence and a sequence specific to the gene of interest, and the PCR-amplified sequence is analyzed. This allows a TCR or BCR repertoire library and a library for gene expression analysis to be prepared and analyzed in parallel.

[0042] In one embodiment, the method according to the present invention is the preparation and analysis of TCR or BCR repertoires at the single-cell level, and is based on a combination of a single-cell analysis device as described in Patent Document 1 and the new library preparation method as described above. Specifically, using a device as described in Patent Document 1, cell capture → cell lysis → mRNA capture → cDNA synthesis by reverse transcription reaction are carried out for each cell in a plurality of micro-reaction chambers. 10 micro-magnetic beads (e.g., Φ: 1 μm) with RT probes immobilized thereon are placed in the micro-reaction chambers. 5 Because they are packed so densely, there are a total of 10 10 There are more than 1000 RT probes available, and it is possible to capture minute amounts of mRNA from a single cell and synthesize cDNA without sample loss.

[0043] The device described in Patent Document 1 is a device having multiple micro-reaction chambers. An example of the configuration of such a device is shown in Figure 8. The micro-reaction chambers of this device are filled with one or more solid phases to which RT probes are immobilized. Each micro-reaction chamber is formed with a through-hole, through which a single cell is captured. The through-hole can be appropriately set depending on the size of the cell to be analyzed, but preferably has a diameter of 10 μm or less. Using such a device, multiple cells are introduced into the micro-reaction chambers so that each micro-reaction chamber corresponds to a single cell. At this time, negative pressure is applied (suction) to the through-holes, thereby capturing a single cell in each through-hole. mRNA eluted from each single cell is captured by the RT probe immobilized on the solid phase of the corresponding micro-reaction chamber. Such devices are known in the art and are not particularly limited. For example, devices described in Patent Document 1, International Publication WO 2016 / 038670, etc. can be used.

[0044] An embodiment of the method of the present invention for preparing a repertoire library at the single-cell level is described with reference to FIG. 2. As shown in FIG. 2(1), the sequence of an RT probe 213 immobilized on a solid phase 206 consists of an amplification sequence 212, a cell identification barcode 211, a UMI 210, and an oligo(dT) 209. The RT probe 213 is captured by the poly(A) tail 203 of a gene (mRNA) 204. By synthesizing cDNA 208 using this RT probe 213 and a solution-phase TSO 105, the cell identification barcode 211, the UMI 210, and the amplification sequence 212 are introduced into the 3' end. A TS sequence 207 is also added to the 5' end of the TCR gene (mRNA) 204. Subsequently, PCR amplification is performed using a FW / RV primer set 214, 215 (corresponding to the TS sequence and the amplification sequence), both of which have phosphate modifications at their 5' ends (FIG. 2(2)). Phosphate groups are added to the 5'-ends of both ends of the obtained PCR amplification product with 100% efficiency. This allows efficient ligation between the TS sequence 207 on the 5' side of the gene and the amplification sequence 212 on the 3' side, resulting in highly efficient formation of circularized DNA ((3) in Figure 2). This process brings the cell identification barcode 211 and UMI 210 into close proximity with the TCR variable region 201. In Example 1, which will be described later, five commercially available types of ligase were used to compare and verify the reaction efficiency (ligation efficiency), confirming that all were above 60%, with the highest being a high value of 72.6% (Figure 4).

[0045] After cDNA is synthesized with high efficiency in (1) of Figure 2, the product is sufficiently amplified by PCR in (2) of Figure 2, and the circularized DNA is formed using the target sample in (3) of Figure 2. The efficiency of this process is 72.6%, which means that there is almost no sample loss of molecules derived from a single cell.

[0046] Next, as shown in Figure 2(4), PCR amplification was performed using FW / RV primer set 216, 217 (corresponding to the 5' and 3' sequences of the C region) designed for the C region 202 of the circularized DNA. This removed the long C region and yielded a short amplification product (several hundred bases) containing both (1) the TCR variable region and (2) the cell identification barcode and UMI (Figure 2(5)). The region excluding the NGS consensus sequences 218, 219 (total 116 bases) at both ends of the library could be sequenced in one go using NGS (Miseq, capable of analyzing up to 600 bases). A library for gene expression analysis was also prepared from the sample amplified with FW / RV primer set 214, 215, which has a phosphate-modified 5' end. NGS data for both TCR repertoire analysis and gene expression analysis were separated using the CT barcode 220, cell identification barcode 211, and UMI 210. By integrating both, it is possible to obtain simultaneous analytical information on TCR repertoire and gene expression at the single cell level.

[0047] This method can be applied not only to single-cell analysis but also to bulk TCR repertoire analysis using multiple cells. As shown in Figure 2(2), gene (mRNA) 204 is amplified by PCR (FW / RV primer set 214, 215 (corresponding to the TS sequence and amplification sequence) with phosphate-modified 5' ends to form circularized DNA (Figure 2(3)). Next, as shown in Figure 2(4), PCR amplification is performed using FW / RV primer set 216, 217, designed for the C region of the circularized DNA, to obtain a short amplification product (several hundred bases) containing both (1) the TCR variable region and (2) the UMI (Figure 2(5)). Libraries for gene expression analysis can also be prepared from samples amplified with FW / RV primer set 214, 215 with phosphate-modified 5' ends. After sequencing these libraries using NGS (Miseq), the NGS data is separated using CT barcode 220 and UMI 210, and by integrating both, simultaneous analysis information on TCR repertoire and gene expression can be obtained efficiently and easily. [Example]

[0048] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0049] [Example 1] In this example, cDNA synthesized with high efficiency from mRNA using the single-cell analysis device described in Patent Document 1 was amplified by PCR using a 5'-phosphorylated primer set (214, 215). The product was then self-ligated with ligase to form circular DNA, bringing the TCR variable region (201) and cell identification barcode (211) / UMI (210) into close proximity. This was then amplified by PCR using a FW / RV primer set (216, 217) designed for the C region (202). This resulted in the preparation of a library for TCR repertoire analysis, in which the TCR variable region (201) and cell identification barcode (211) / UMI (210) are contained within a short fragment (221) of several hundred bases. Furthermore, a library for gene expression analysis can also be prepared from the same cDNA amplified by PCR using a 5'-phosphorylated primer set (214, 215). This library can then be subjected to NGS analysis, enabling simultaneous acquisition of TCR repertoire and gene expression data from a single cell. Because library preparation is performed while the cell identification barcode / UMI remains inserted into the 3' end of the gene (mRNA), the process of inserting the cell identification barcode / UMI into the 5' end (which has low reaction efficiency), as employed in conventional technology, is unnecessary, making this an example of achieving highly sensitive analysis that avoids sample loss.

[0050] A flowchart of this method is shown in Figure 3. The analysis method based on this example will be described below in the following order: 1. Cell capture using a single-cell analysis device (equipped with 16 VFACs (801)) and reverse transcription and TS-PCR amplification after cell lysis, 2. Preparation of a library for TCR repertoire analysis, and 3. Preparation of a library for gene expression analysis.

[0051] 1. Cell capture using a single-cell analysis device, cell lysis, reverse transcription, and TS-PCR amplification Figure 8 shows an example of a single-cell analysis device. The chip (VFACs (801)) installed in the single-cell analysis device is made of PDMS. The through-holes (802) at the top of the micro-reaction chambers for capturing cells (803) are laser-machined to an average diameter of 3 μm. The micro-reaction chambers (805) filled with beads are cylindrical with an inner diameter of 75 μm and a depth of 70 μm. An oligo(dT) sequence-containing RT probe (SEQ ID NO: 1) (811 and 213) for capturing mRNA is immobilized on the beads (1 μm diameter (804)). As described in Patent Document 1, this RT probe contains a cell identification barcode (SEQ ID NO: 2) (211), a molecular identification barcode UMI (SEQ ID NO: 3) (210), and an amplification sequence (SEQ ID NO: 4) (212). Each device is equipped with 16 VFACs (801), making it possible to acquire NGS data (TCR repertoire data, gene expression data) for up to 1,600 single cells. The processing method from reverse transcription reaction to TS-PCR amplification after cell dispensing is shown below.

[0052] Add 4 μL of phosphate-buffered saline (PBS) solution (0.8 U / μL RNase OUT, 0.1% Tween 20) to each of the 16 VFACs (801) mounted on the single-cell analysis device, then apply pump suction (90 kPa) (807) from below the VFACs (801). This allows for pre-washing. Next, add 4 μL of PBS solution (0.8 U / μL RNase OUT, 0.1% Tween 20, 0.2% BSA) to each VFAC, then quickly add 1 μL of T cell suspension (80 cells / μL, mouse) (803) to each VFAC, and apply pump suction (90 kPa) (807) from below the VFACs. This allows individual cells (803) to be captured on the VFACs (801). Next, 8 μL of cell wash buffer (100 mM Tris (pH 8.0), 500 mM NaCl, 5 mM DTT, 0.4 U / μL RNase OUT, 0.1% Tween 20) is added, followed by pump suction (90 kPa) (807) to recapture the small number of cells remaining on the surface of the VFACs. 1 μL of cell lysis buffer (100 mM Tris (pH 8.0), 500 mM NaCl, 10 mM EDTA, 1% SDS, 5 mM DTT, 1.33 U / μL RNase OUT) is added to lyse the cells (808, 809), and the eluted mRNA (810) is captured by the bead-immobilized RT probe (811). After the reaction, the reagent is removed by pump suction (90 kPa) (807). After adding 8 μL of lysis wash buffer (100 mM Tris (pH 8.0), 500 mM NaCl, 5 mM DTT, 0.4 U / μL RNase OUT, 1% Tween 20), pump aspiration (90 kPa) is performed to remove residual reagents from the Cell Lysis Buffer that may cause enzyme reaction inhibition. This procedure is repeated twice. In this step, even if a series of pump aspirations (807) are performed at a force of 90 kPa, cell-derived mRNA (approximately 10 6 It has been confirmed that there is no loss of 810 molecules / cells. Both the Cell Lysis buffer and the Lysis wash buffer contain a high concentration of NaCl (500 mM) and a sufficient amount (1.5x10 10Since the RT probe (811) of the molecule is immobilized on the magnetic beads (804), the trace amount of mRNA in the cell is efficiently captured.

[0053] 4.5 μL of reverse transcription reagent (1x SSIV Buffer (ThermoFisher), 1.25 mM TSO (SEQ ID NO: 5), 5 mM DTT, 2 mM dNTPs, 3.5 U / μL RNase OUT (ThermoFisher), 10 Units / μL SS IV (ThermoFisher), 0.5% Tween 20) was added to the top of each VFAC. The opening on the top of the flow cell device was sealed (ThermoFisher, Optical Adhesive Film) and the device was incubated in a preheated incubator at 50°C for 60 minutes. This reverse transcription reaction synthesized cDNA containing the TS sequence (207) at the 3' end and the cell identification sequence (SEQ ID NO: 3) (211), UMI (SEQ ID NO: 2) (210), and amplification sequence (SEQ ID NO: 4) (212) at the 5' end. After incubating at room temperature for 5 minutes, remove the seal from the device and aspirate the reverse transcription reagents using a pump (807). Add 100 μL of 50 mM Tris Buffer (pH 8.0, 0.1% Tween 20) to each of the 16 PCR tubes, and remove the VFAC (801) and membrane from the flow cell device. Place a neodymium magnet close to the bottom of the tube to thoroughly mix the beads (804) packed in the VFAC's microreaction chambers into the buffer. Wash the beads twice with 50 μL of 50 mM Tris Buffer (pH 8.0, 0.1% Tween 20). Add 15 μL of Exonuclease I reaction solution (1x Exonuclease I buffer, 0.067 Unit / μL Exonuclease I) prepared on ice to the beads, mix, and incubate at 37°C for 15 minutes. After capturing the beads by bringing a neodymium magnet close to the beads, the supernatant is removed and the beads are washed three times with 50 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0).The beads were suspended in 1 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) and mixed with 9 μL of TS-PCR PCR reagents (1x Gflex PCR buffer, 1 μM TS primer (5'-terminal phosphorylation, SEQ ID NO: 6) (214), 1 μM amplification primer (5'-terminal phosphorylation, SEQ ID NO: 7) (215), 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) prepared on ice. PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 18 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 3 minutes, followed by a 2-minute 68°C period followed by a constant temperature of 4°C. For each of the 16 samples, beads were captured with a neodymium magnet, and 10 μL of the supernatant containing the TS-PCR amplified product was transferred to a separate tube. The beads were washed with 40 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0), and the supernatant was mixed with the amplified product to a total volume of 50 μL. 35 μL of Ampure XP, equivalent to 0.7x the sample volume, was added to each sample, mixed, and purified according to the manufacturer's recommended protocol. Finally, the product was eluted with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0054] 2. Preparation of libraries for TCR repertoire analysis A portion of the TS-PCR amplification product was used to prepare a library for TCR repertoire analysis. The self-ligation step (formation of circular DNA) using ligase performed here is a critical step in the method of the present invention. To confirm whether this step results in sample loss, the reaction efficiency of this self-ligation step was evaluated using five commercially available ligases (Table 1).

[0055] [Table 1]

[0056] The results showed that all were highly efficient, exceeding 60% (Figure 4). With the most effective ligase (Quick Ligation kit, NEB), it was confirmed that circularized DNA could be formed by self-ligation with an efficiency of 72.6%. Therefore, after synthesizing cDNA from mRNA with high efficiency as described above, the product sufficiently amplified by TS-PCR could be self-ligated (formed circularized DNA) with an efficiency of 70%, which indicates that the sample was prepared with almost no loss from the initial mRNA sample. The detailed method for library preparation for TCR repertoire analysis is described below.

[0057] For each of the 16 samples, 5 μL of the TS-PCR amplified product was transferred to a separate tube and mixed with 5 μL of Blunt / TA Ligase Master Mix (NEB) to make a 10 μL solution. The mixture was then incubated at room temperature for 10 minutes. This allowed the TS-PCR amplified product, which had both 5'-ends phosphorylated, to self-ligate efficiently to form circular DNA. The mixture was then incubated at 65°C for 10 minutes to inactivate any remaining ligase. For each of the 16 samples, 3 μL of the heat-treated sample was transferred to a separate tube and mixed with 27 μL of TRA gene PCR reagent (1) (1x Gflex PCR buffer, 1 μM TCRα-C1-FW primer (SEQ ID NO: 8) (216), 1 μM TCRα-C1-RV primer (SEQ ID NO: 9) (217), and 0.07 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. For each of the 16 samples, 3 μL of the heat-treated sample was transferred to a separate tube and mixed with 27 μL of PCR reagent (1) for the TRB gene (1) (1x Gflex PCR buffer, 1 μM TCRβ-C1-FW primer (SEQ ID NO: 10) (216), 1 μM TCRβ-C1-RV primer (SEQ ID NO: 11) (217), and 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. The C1 primer set (216, 217) for the TRA and TRB genes used here was designed using the C region sequence (202) of each gene. Specifically, the C1-FW primer (216) utilizes sequences near the 3' end of the C region of each gene (mRNA) (downstream near the poly(A) tail), and the C1-RV primer (217) utilizes sequences near the 5' end of the C region (upstream near the variable region). PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 16 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 30 seconds, followed by 68°C for 2 minutes, followed by a constant temperature of 4°C. Add 20 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) to each of the 16 samples and mix to make a total of 50 μL.Add 35 μL of Ampure XP (equivalent to 0.7x the sample volume) to each sample, mix, and purify according to the manufacturer's recommended protocol. Finally, elute with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0058] Nested PCR was performed to enhance detection sensitivity (sequence specificity) and to add consensus sequences (SEQ ID NOs: 109 and 110) for NGS and CT barcodes (7-base known sequences, SEQ ID NO: 108 is an example) that identify VFACs. For each of the 16 samples, 3 μL of the TRA gene amplification product was transferred to a separate tube and mixed with 27 μL of TRA gene PCR reagent (2) (1x Gflex PCR buffer, 1 μM TCRα-C2-FW primer (SEQ ID NO: 12), 1 μM TCRα-C2-CT1_RV primer (a primer containing a different CT barcode for each VFAC; SEQ ID NO: 13 is an example), and 0.075 Units / μL Tks Gflex DNA polymerase (Takarabio)) on ice. For each of the 16 samples, 3 μL of the TRB gene amplification product was transferred to a separate tube and mixed with 27 μL of TRB gene PCR reagent (2) (1x Gflex PCR buffer, 1 μM TCRβ-C2-FW primer (SEQ ID NO: 14), 1 μM TCRβ-C2-CT1_RV primer (a primer containing a different CT barcode for each VFAC; SEQ ID NO: 15 is an example), and 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. The C2-FW primer for the TRA and TRB genes used in this nested PCR utilizes a sequence further 3' from the C region (downstream near the poly(A) tail) than the C1-FW primer, while the C2-RV primer utilizes a sequence further 5' from the C region (upstream near the variable region) than the C1-RV primer. PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 16 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 30 seconds, followed by 2 minutes at 68°C, followed by a constant temperature of 4°C. 20 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) was added to each of the 16 samples and mixed to a total volume of 50 μL. 35 μL of Ampure XP, equivalent to 0.7 times the sample volume, was added to each sample and mixed, and purification was performed according to the manufacturer's recommended protocol. Finally, elution was performed with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0059] One microliter of PCR amplification product was analyzed by chip electrophoresis to determine the product size (bp) and concentration (pmol / L), followed by next-generation sequencing (NGS) analysis (Illumina, Miseq). The data from the NGS analysis was separated into barcode sequences (CT barcode (SEQ ID NO: 108) (220), cell identification barcode (SEQ ID NO: 3) (211), UMI (molecular identification barcode) (210), SEQ ID NO: 2), allowing for the separation and acquisition of repertoire data for each sample, cell, and molecule. Figure 5A shows the electrophoresis results for the library obtained in this example. Both TRA and TRB are short, consisting of several hundred bases. Both of these sequences contain the 58-base NGS consensus sequence 1 (SEQ ID NO: 110) (218) and the NGS consensus sequence 2 (SEQ ID NO: 109) (219), both of which are 58 bases long. The inner regions, excluding these 116-base consensus sequences, can be sequenced using NGS (Miseq). Because the library size is short, we used an NGS system (Miseq) capable of analyzing 600 bases, which enabled us to determine the sequences of the variable region (201), CT barcode (SEQ ID NO: 108) (220), cell identification barcode (SEQ ID NO: 3) (211), and UMI (molecular identification barcode) (210) (SEQ ID NO: 2) in a single step. Furthermore, the NGS data obtained confirmed that approximately 99% of the total reads mapped to the TRA and TRB genes (Figure 5B).

[0060] 3. Library preparation for gene expression analysis A portion of the TS-PCR amplified product will be used to prepare a library for expression analysis of 44 cancer immune-related genes. The detailed method is shown below.

[0061] For each of the 16 samples, 1 μL of the TS-PCR amplified product was transferred to a separate tube and mixed with 9 μL of 1st Multiplex PCR reagent (1x Gflex PCR buffer, 0.2 μM 44plex FW primer mix (SEQ ID NOs: 16-59), 2 μM amplification sequence_RV primer (SEQ ID NO: 7), 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 7 cycles of 98°C for 10 seconds, 58°C for 3 minutes, and 68°C for 25 seconds, followed by a 2-minute incubation at 68°C and a constant temperature of 4°C. 40 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) was added to each of the 16 samples and mixed to a total volume of 50 μL. 35 μL of Ampure XP, equivalent to 0.7x the sample volume, was added to each sample, mixed, and purified according to the manufacturer's recommended protocol. Finally, elution was performed with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20). Next, a second multiplex PCR was performed to enhance detection sensitivity (sequence specificity) and to partially add a consensus sequence for NGS. For each of the 16 samples, 7 μL of the first multiplex PCR amplified product was transferred to a separate tube and mixed with 13 μL of second multiplex PCR reagents (1x Multiplex PCR Plus (QIAGEN), 0.3 μM 2nd R2SP-added 44plex FW primer mix (SEQ ID NOs: 60-103), and 3 μM amplification sequence_RV primer (SEQ ID NO: 7)) on ice. PCR amplification was performed under the following temperature conditions: heat inactivation at 95°C for 5 minutes, followed by three cycles of 95°C for 30 seconds, 61°C for 5 minutes, and 72°C for 30 seconds; three cycles of 95°C for 30 seconds, 59°C for 5 minutes, and 72°C for 30 seconds; three cycles of 95°C for 30 seconds, 57°C for 5 minutes, and 72°C for 30 seconds; three cycles of 95°C for 30 seconds, 55°C for 5 minutes, and 72°C for 30 seconds; and a constant temperature of 4°C after 2 minutes at 72°C. For each of the 16 samples, add 30 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) and mix to a total volume of 50 μL.Add 35 μL of Ampure XP (equivalent to 0.7x the sample volume) to each sample, mix, and purify according to the manufacturer's recommended protocol. Finally, elute with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0062] Third PCR was performed to add consensus sequences for NGS analysis (SEQ ID NOs: 109 and 110) and a CT barcode (a 7-base known sequence, SEQ ID NO: 108) for identifying VFACs. For each of the 16 samples, 8 μL of the second PCR product was transferred to a separate tube and mixed with 22 μL of third PCR reagent (1x Gflex PCR buffer, 0.23 μM P5_R1SP_CT_amplification sequence_RV primer (SEQ ID NO: 104), 0.23 μM P7_R2SP_FW primer (SEQ ID NO: 105), 0.01 μM P5_RV primer (SEQ ID NO: 106), 0.01 μM P7_FW primer (SEQ ID NO: 107), and 0.045 Units / μL Tks Gflex DNA polymerase (Takarabio)) on ice. PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by two cycles of 98°C for 10 seconds, 61°C for 1 minute, and 68°C for 25 seconds; two cycles of 98°C for 10 seconds, 59°C for 1 minute, and 68°C for 25 seconds; two cycles of 98°C for 10 seconds, 57°C for 1 minute, and 68°C for 25 seconds; two cycles of 98°C for 10 seconds, 55°C for 1 minute, and 68°C for 25 seconds; and a 4°C constant temperature after 68°C for 2 minutes. For each of the 16 samples, add 20 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) and mix to a total volume of 50 μL. Add 35 μL of Ampure XP (equivalent to 0.7x the sample volume) to each sample, mix, and purify according to the manufacturer's recommended protocol. Finally, elute with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0063] One microliter of the 3rd PCR amplified product was analyzed by chip electrophoresis to determine the product size (bp) and concentration (pmol / L), followed by next-generation sequencing (NGS) analysis (Illumina, Miseq). The data from the NGS analysis was separated by barcode sequence (CT barcode (SEQ ID NO: 108), cell identification barcode (SEQ ID NO: 3), and UMI (molecular identification barcode, SEQ ID NO: 2)) and mapped to sequences published in a public database of 44 genes. This allowed the number of mRNA molecules to be counted and gene sequence data for each cell to be obtained. The results are shown in Figure 6. Figure 6 shows the number of mRNA molecules for each gene. After separating the data for each cell identification barcode sequence, it was merged with the results of the TCR repertoire analysis described above to extract gene expression data and TCR repertoire data at the single-cell level.

[0064] [Example 2] This example demonstrates a simple and efficient method for preparing libraries for TCR repertoire analysis from mRNA in the initial sample, even when using bulk samples (multiple T cells) such as those used in MRD analysis, rather than single cells. Because the library is prepared while the UMI remains at the 3' end of the gene (mRNA), the process of introducing the UMI at the 5' end (which has low reaction efficiency) employed in conventional techniques is unnecessary, avoiding sample loss and enabling highly sensitive analysis. A flowchart of this method is shown in Figure 7. The analysis method based on this example is described below in the following order: 1. Reverse transcription and TS-PCR amplification using multiple cells, 2. Ligase-based self-ligation (formation of circular DNA), and 3. PCR amplification using FW and RV primer sets designed for the C region (5' and 3' ends).

[0065] 1. Reverse transcription and TS-PCR amplification using multiple cells 1 mL of T cell suspension (10 5The cells were lysed by centrifugation (200g, 5 min, 4°C) of the lysed cells (100mM Tris (pH 8.0), 500mM NaCl, 10mM EDTA, 1% SDS, 5mM DTT, 1.33U / µL RNase OUT) and 25µL of RT probe-immobilized magnetic beads (Φ1µm, 10 7 Cell lysis buffer and lysis wash buffer contain a high concentration of 500 mM NaCl and a sufficient amount (1.5x10) of NaCl (SEQ ID NO: 1) (213) and mix well. Incubate at room temperature for 2 minutes. After capturing the beads with a neodymium magnet, remove the supernatant and wash twice with 50 μL of lysis wash buffer (100 mM Tris (pH 8.0), 500 mM NaCl, 5 mM DTT, 0.4 U / μL RNase OUT, 1% Tween 20). Both the cell lysis buffer and lysis wash buffer contain a high concentration of NaCl (500 mM) and contain a sufficient amount (1.5x10) of NaCl. 10 The RT probe (SEQ ID NO: 1) (213) of the mRNA (molecules) is immobilized on magnetic beads, allowing efficient capture of the trace mRNA (204) in cells. 50 μL of reverse transcription reagent (1x SSIV Buffer (ThermoFisher), 1.25 mM TSO, 5 mM DTT, 2 mM dNTPs, 3.5 U / μL RNase OUT (ThermoFisher), 10 Units / μL SS IV (ThermoFisher), 0.5% Tween 20) is added and incubated at 50°C for 60 minutes. This reverse transcription reaction synthesizes cDNA containing a TS sequence (207) at the 3' end and a cell identification sequence (SEQ ID NO: 3) (211), a UMI (SEQ ID NO: 2) (210), and an amplification sequence (SEQ ID NO: 4) (212) at the 5' end.

[0066] After incubating at room temperature for 5 minutes, capture the magnetic beads with a neodymium magnet and remove the supernatant. Wash the beads twice with 100 μL of 50 mM Tris Buffer (pH 8.0, 0.1% Tween 20). Mix with 30 μL of Exonuclease I reaction solution (1x Exonuclease I buffer, 0.067 Unit / μL Exonuclease I) prepared on ice and incubate at 37°C for 15 minutes. Capture the beads by bringing the neodymium magnet close to the beads. Remove the supernatant and wash the beads three times with 50 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0). 30 μL of TS-PCR PCR reagent (1x Gflex PCR buffer, 1 μM TS_FW primer (5'-end phosphorylation, SEQ ID NO: 6) (214), 1 μM amplification sequence_RV primer (5'-end phosphorylation, SEQ ID NO: 7) (215), and 0.075 Units / μL Tks Gflex DNA polymerase (Takarabio)) was mixed on ice. PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 9 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 3 minutes, followed by 68°C for 2 minutes and a constant temperature of 4°C. After capturing the beads with a neodymium magnet, 30 μL of the supernatant containing the TS-PCR amplification product was transferred to a separate tube. The beads were washed with 20 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0), and the supernatant was mixed with the amplification product to a total volume of 50 μL. Add 40 μL of Ampure XP (equivalent to 0.7x the sample volume) to the sample, mix, and purify according to the manufacturer's recommended protocol. Finally, elute with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0067] 2. Self-ligation using ligase (formation of circular DNA) A 5 μL aliquot of the TS-PCR amplified product was transferred to a separate tube and mixed with 5 μL of Blunt / TA Ligase Master Mix (NEB) to make a 10 μL solution. This was then incubated at room temperature for 10 minutes. This allowed the TS-PCR amplified product, which has phosphorylated 5' ends at both ends, to self-ligate efficiently and form circular DNA. The sample was then incubated at 65°C for 10 minutes to inactivate any remaining ligase.

[0068] 3. PCR amplification using the FW / RV primer set (216, 217) designed for the C region (5' and 3' ends) A 3 μL aliquot of the heat-treated sample was placed in a separate tube and mixed with 27 μL of PCR reagent (1) for the TRA gene (1) (1x Gflex PCR buffer, 1 μM TCRα-C1-FW primer (SEQ ID NO: 8) (216), 1 μM TCRα-C1-RV primer (SEQ ID NO: 9) (217), 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. Another 3 μL aliquot of the heat-treated sample was placed in a separate tube and mixed with 27 μL of PCR reagent (1) for the TRB gene (1) (1x Gflex PCR buffer, 1 μM TCRβ-C1-FW primer (SEQ ID NO: 10) (216), 1 μM TCRβ-C1-RV primer (SEQ ID NO: 11) (217), 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. The C1 primer set for the TRA and TRB genes used here was designed using the C region sequences of each gene (202). Specifically, the C1-FW primer (216) utilizes sequences near the 3' end of the C region of each gene (mRNA) (downstream near the poly(A) tail), and the C1-RV primer utilizes sequences near the 5' end of the C region (upstream near the variable region) (217). PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 16 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 30 seconds, followed by 2 minutes at 68°C, followed by a constant temperature of 4°C. Each of the 16 samples was mixed with 20 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) to a total volume of 50 μL. Add 35 μL of Ampure XP (equivalent to 0.7x the sample volume) to each sample, mix, and purify according to the manufacturer's recommended protocol. Finally, elute with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0069] Nested PCR was performed to enhance detection sensitivity (sequence specificity) and to add consensus sequences (SEQ ID NOs: 109 and 110) for NGS and a CT barcode (SEQ ID NO: 108) for sample identification. 3 μL of the TRA gene amplified product was transferred to a separate tube and mixed with 27 μL of TRA gene PCR reagent (2) (1x Gflex PCR buffer, 1 μM TCRα-C2-FW primer (SEQ ID NO: 12), 1 μM TCRα-C2-CT1_RV primer (a primer containing a different CT barcode (220) for each sample; SEQ ID NO: 13 is an example), and 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. Transfer 3 μL of the TRB gene amplified product to a separate tube and mix with 27 μL of TRB gene PCR reagent (2) (1x Gflex PCR buffer, 1 μM TCRβ-C2-FW primer (SEQ ID NO: 14), 1 μM TCRβ-C2-RV primer (a primer containing a different CT barcode (220) for each sample was used; SEQ ID NO: 15 is an example), and 0.075 Unit / μL Tks Gflex DNA polymerase (Takarabio)) on ice. The C2-FW primer for the TRA and TRB genes used in this nested PCR utilizes a sequence further 3' from the C region (downstream near the poly(A) tail) than the C1-FW primer (216), and the C2-RV primer utilizes a sequence further 5' from the C region (upstream near the variable region) than the C1-RV primer (217). PCR amplification was performed under the following temperature conditions: heat inactivation at 94°C for 1 minute, followed by 16 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 30 seconds, followed by 2 minutes at 68°C, followed by a constant temperature of 4°C. 20 μL of wash buffer (0.1% Tween 20, 10 mM Tris, pH 8.0) was added and mixed to a total volume of 50 μL. 35 μL of Ampure XP, equivalent to 0.7 times the sample volume, was added and mixed to each sample, and purification was performed according to the manufacturer's recommended protocol. Finally, elution was performed with 40 μL of wash buffer (10 mM Tris (pH 8.0), 0.1% Tween 20).

[0070] One microliter of PCR amplification product was analyzed by chip electrophoresis to determine the product size (bp) and concentration (pmol / L), followed by next-generation sequencing (NGS) analysis (Illumina, Miseq). The data from the NGS analysis was separated into barcode sequences (chip barcode (220), UMI (molecular identification barcode) (210)), allowing for the acquisition of separate repertoire data for each sample and molecule. The electrophoresis results for the library obtained in this example were similar to those shown in Figure 5(A) of Example 1, confirming that both TRA and TRB are short, consisting of several hundred bases. Both of these sequences contain the 58-base NGS consensus sequence 1 (SEQ ID NO: 110) (218) and NGS consensus sequence 2 (SEQ ID NO: 109) (219), both of which are 58 bases long. The inner regions excluding these 116-base consensus sequences can be sequenced using NGS (Miseq). Because the library size is short, the sequences of the variable region (201), CT barcode (SEQ ID NO: 108) (220), and UMI (molecular identification barcode) (210) (SEQ ID NO: 2) can be determined in one go using an NGS system (Miseq) capable of analyzing 600 bases. Furthermore, the NGS data obtained was similar to the results in Example 1 (Figure 5(B)), confirming that approximately 99% of the total reads were mapped to the TRA and TRB genes.

[0071] [Array Description] All sequences shown below are in the 5' to 3' direction. SEQ ID NO: 1: RT probe (one of 100 types of cell identification barcodes, N = A, G, C, N = A, G, C, T) CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGCGTACNNNNNNNTTTTTTTTTTTTTTTTVN SEQ ID NO: 2: UMI (molecular identification barcode, N=A, G, C, T) NNNNNNN SEQ ID NO: 3: Cell identification barcode (8-base known sequence. Shown is an example of 100 known sequences) TCGCGTAC SEQ ID NO: 4: Amplification sequence (reverse primer) CCATCTCATCCCTGCGTGTCT SEQ ID NO: 5: TSO (Template Switching Oligo, rG = RNA base) AAGCAGTGGTATCAACGCAGAGTACATrGrGrG SEQ ID NO: 6: TS_FW primer (5'-end phosphorylation modification, FW primer) AAGCAGTGGTATCAACGCAGA SEQ ID NO: 7: Amplification sequence_RV primer (5'-end phosphorylation modification, RV primer) CCATCTCATCCCTGCGTGTCT SEQ ID NO: 8: TCRα-C1-FW primer CAGCTCCCTCACTGCTGCT SEQ ID NO: 9: TCRα-C1-RV primer TGCTCTTGGAATCCATAGCTT SEQ ID NO: 10: TCRβ-C1-FW primer GTCTTGTCTGCCACCATCCTC SEQ ID NO: 11: TCRβ-C1-RV primer TGGACCTCCTTGCCATTCACC SEQ ID NO: 12: TCRα-C2-FW primer (NGS consensus sequence P7R2SP added) CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATAATTATTCTGCTGTCCATG SEQ ID NO: 13: TCRα-C2-CT1_RV primer (NGS common sequence P5R1SP and CT1 added) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACATATGTGTACCAGTTAAAAGATC SEQ ID NO: 14: TCRβ-C2-FW primer (NGS consensus sequence P7R2SP added) CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAACATGTTCTAGGACGGCCT SEQ ID NO: 15: TCRβ-C2-CT1_RV primer (NGS common sequence P5R1SP, and CT1 added) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACATAAGGTCTCCTTGTTTGAGCCA SEQ ID NO: 16: Acaca gene_1st FW primer TCTCTGGCCTCCACTTTTGCT SEQ ID NO: 17: Batf gene_1st FW primer GAGGTGGTATACAGTGCCCAT SEQ ID NO: 18: Bcl2 gene_1st FW primer AGGATTGATGGCAGATTCAGT SEQ ID NO: 19: Bcl-6 gene_1st FW primer GACTATTTTAAGTATTGCGTCTGT SEQ ID NO: 20: Btla gene_1st FW primer CCCTTTTCTATTGCACTCCAC SEQ ID NO: 21: Cd160 gene_1st FW primer ATGAACAGAGAAGTCATTTGCC SEQ ID NO: 22: Cd244 gene_1st FW primer GCCAGATGTCAGTCTTGTTCAC SEQ ID NO: 23: Cd3e gene_1st FW primer GCTAAGCCCTTTCCTACAGC SEQ ID NO: 24: Cd4 gene_1st FW primer TTCTCTTAGAGTGAGGCTGGG SEQ ID NO: 25: Cd8a gene_1st FW primer TGGAGAACATTCCTTAGCACCC SEQ ID NO: 26: Cd8b1 gene_1st FW primer CACAGAGTGGCTGAAGAACCA SEQ ID NO: 27: Ctla4 gene_1st FW primer GGCTCAGTTGCATAAACCGAT SEQ ID NO: 28: Cxcr3 gene_1st FW primer CGCAGCCCAAGTCCTAACACA SEQ ID NO: 29: Eomes gene_1st FW primer TTGTTGTAGGGCTGGCTCTGT SEQ ID NO: 30: Fas gene_1st FW primer GAGAGAGCCTGCCACCCAT SEQ ID NO: 31: Fasl gene_1st FW primer TACAAGGGTGAGAAAGGAGGC SEQ ID NO: 32: Foxp3 gene_1st FW primer CTATGCCCCTATAAGACCACCCT SEQ ID NO: 33: Gzmb gene_1st FW primer GCAGGCCAATGGAACACCTC SEQ ID NO: 34: Havcr2 gene_1st FW primer AATATTCTTTGGGAGGACAGTCG SEQ ID NO: 35: Hif-1a gene_1st FW primer TGCAGTATGAATGGAGTAAGTGA SEQ ID NO: 36: Icos gene_1st FW primer CCTCTAGTCTTTGGTCTGCAT SEQ ID NO: 37: Ifng gene_1st FW primer CTGACTAATTAGCCAAGACTGTG SEQ ID NO: 38: Il10 gene_1st FW primer TTTAAGCTGTTTCCATTGGGG SEQ ID NO: 39: Il17a gene_1st FW primer AGAATTGTCTGCCCTCCACA SEQ ID NO: 40: Il2rb gene_1st FW primer ACGAAGAGGTCCTGATAAGACTG SEQ ID NO: 41: Il2rg gene_1st FW primer TCCTAAGTGACGCTAACCTCC SEQ ID NO: 42: Il7r gene_1st FW primer GTCACAATTTTGAATCCCTGCTG SEQ ID NO: 43: Klrg1 gene_1st FW primer TCATGTGCTCACTCCTGCTT SEQ ID NO: 44: Lag3 gene_1st FW primer GTACGCCGCAGAGTCTAGCTCA SEQ ID NO: 45: Mki67 gene_1st FW primer AAACTAGAGTCGCTTTGGACAG SEQ ID NO: 46: Pdcd1 gene_1st FW primer TGATGAAGACTTGAAAAGCTCCT SEQ ID NO: 47: Prdm1 gene_1st FW primer GCAGCCCCAACAAGACTGACA SEQ ID NO: 48: Prf1 gene_1st FW primer CCTACCATGCCAAGTGTCTGC SEQ ID NO: 49: Rorc gene_1st FW primer TCCTGAGACTTTTCTGCCTTTC SEQ ID NO: 50: Runx3 gene_1st FW primer CAGTGAGCACAGGCAGCTGGG SEQ ID NO: 51: Slc2a1 gene_1st FW primer CCTCGGCACCATAGGGGTC SEQ ID NO: 52: Tbp gene_1st FW primer GCAGCACTACTGTGAGTTGCTT SEQ ID NO: 53: Tbx21 gene_1st FW primer GGTCGGGGTGGGGAGTCCAG SEQ ID NO: 54: Tgfb gene_1st FW primer TGCCAACTTCTGTCTGGGACCCT SEQ ID NO: 55: Tigit gene_1st FW primer CTGTGCTGGGACTCATTTGCT SEQ ID NO: 56: Tnf gene_1st FW primer GAGGCTGGATAAGATCTCAGG SEQ ID NO: 57: Tnfrsf9 gene_1st FW primer TTTGAGGGGAATCTTCAGAGC SEQ ID NO: 58: Vsir gene_1st FW primer TGCCTTGGACTCAGGAAGGGAA SEQ ID NO: 59: Zeb2 gene_1st FW primer GAGACATTTGTGATGTTGGCTGT SEQ ID NO: 60: Acaca gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCCGGTGGTATGAAAGCTG SEQ ID NO: 61: Batf gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCCTTCCACCAGCCTCACATCA SEQ ID NO: 62: Bcl2 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTCCTTTGGAGACAGGGGTTCTCTA SEQ ID NO: 63: Bcl-6 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTGTCTAAAATGCCTCCGTGT SEQ ID NO: 64: Btla gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTCTCATTGAACCTGGAGCTCTG SEQ ID NO: 65: Cd160 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAACACCACATCCAAATACAGGG SEQ ID NO: 66: Cd244 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTCATCCCTGTACATGCCTAG SEQ ID NO: 67: Cd3e gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTATTCCCTTCCCGGCCCAT SEQ ID NO: 68: Cd4 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTGCAGAGCCTGGGAAGTGGA SEQ ID NO: 69: Cd8a gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTAAAGAGGCAGACTAGACGTGG SEQ ID NO: 70: Cd8b1 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACTGGGCCTTGGACAGTGG SEQ ID NO: 71: Ctla4 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATGGTGATGGAGTGTAAACTGGG SEQ ID NO: 72: Cxcr3 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGTGCTTGTCCTCCTTGTAGTTGGG SEQ ID NO: 73: Eomes gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAATTTGGTGCTTTGGCGTTGTAA SEQ ID NO: 74: Fas gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTGACATTGGCAACTCCTGGTG SEQ ID NO: 75: Fasl gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGTGTCTCATTGGCACCATC SEQ ID NO: 76: Foxp3 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAACAGTGCACCTAAAGGACTCA SEQ ID NO: 77: Gzmb gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACCATGCTGTGACAACCCAACTGA SEQ ID NO: 78: Havcr2 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACCTCAGTTTCATCTGGCTT SEQ ID NO: 79: Hif-1a gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCTGTTCCCATTAGCAGGTGA SEQ ID NO: 80: Icos gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCTTCCCCTGGCTTCTAGGATGTATG SEQ ID NO: 81: Ifng gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATTGCGGGGTTGTATCTGGG SEQ ID NO: 82: Il10 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTATATGATGGGAGGGGTTCTTC SEQ ID NO: 83: Il17a gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGGTGTAAAGGGGTCCCAA SEQ ID NO: 84: Il2rb gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTAGAAGGAGGGGTTGGG SEQ ID NO: 85: Il2rg gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAATGCTCACTGCCTTCCCCT SEQ ID NO: 86: Il7r gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATACTGTGGTTGGGTGCCTT SEQ ID NO: 87: Klrg1 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTGTGCCTGAGCTGTAGGGA SEQ ID NO: 88: Lag3 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACCTTAAAGGAGGCCATCTCGT SEQ ID NO: 89: Mki67 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACCTAAGCCAAGGGTAACTCG SEQ ID NO: 90: Pdcd1 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCGTGAGCGTATCTGCTGTCC SEQ ID NO: 91: Prdm1 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAAAGCATGACCTGACATTGACACC SEQ ID NO: 92: Prf1 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGCAGTGGGGCTGTGTGGTAA SEQ ID NO: 93: Rorc gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCTGCCTGTTTTCTGGGAC SEQ ID NO: 94: Runx3 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAAGGGTCCTTATTTGCAAGGCTAC SEQ ID NO: 95: Slc2a1 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTAGGATTCGCCCATTCCTGTC SEQ ID NO: 96: Tbp gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTGCTCTGTGCTGCTACTTGGG SEQ ID NO: 97: Tbx21 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCAAACAGAGAAGACTCAGGTGAC SEQ ID NO: 98: Tgfb gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACCCTGCCCCTATATTTGGAG SEQ ID NO: 99: Tigit gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTATAGAAAGTGGCCTTGGGAG SEQ ID NO: 100: Tnf gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCAATGCACAGCCTTCCTCACA SEQ ID NO: 101: Tnfrsf9 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTGCTTCTCCTGTTTCCTC SEQ ID NO: 102: Vsir gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTCCCAAAAGATTTCCACGTGTGT SEQ ID NO: 103: Zeb2 gene_2nd FW primer (NGS consensus sequence R2SP added) GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAATCTTTGTTACCTTCGCTGTGA SEQ ID NO: 104: P5_R1SP_CT1_amplification sequence_RV primer AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACATACCATCTCATCCCTGCG SEQ ID NO: 105: P7_R2SP_FW primer CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGT SEQ ID NO: 106: P5_RV primer AATGATACGGCGACCACCGAGATCTACAC SEQ ID NO: 107: P7_FW primer CAAGCAGAAGACGGCATACGAGAT SEQ ID NO: 108: CT barcode (7-base known sequence, showing CT1, an example of 16 types) TGACATA SEQ ID NO: 109: P5-R1SP (NGS consensus sequence) AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT SEQ ID NO: 110: P7-R2SP (consensus sequence for NGS) CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT [Explanation of symbols]

[0072] 101, 201: Variable region (VDJ) of TCR gene (or BCR gene) 102, 202: C region (constant region) of TCR gene (or BCR gene) 103, 203: PolyA tail 104, 204: TCR gene (or BCR gene) mRNA 105, 205: TSO(Template Switching Oligo) 106, 206: Solid phase carrier (magnetic beads, etc.) 107, 207: TS sequence (introduced into the cDNA) 108, 208: cDNA synthesized by reverse transcription 109, 209: oligo(dT) 110, 210: UMI (molecular identification barcode) 111, 211: Cell identification barcode 112, 212: Amplification sequences 113, 213: RT probe 114: TS-FW Primer 115: Amplification sequence - RV primer 116: NGS consensus sequence 1 (P7R2SP) 117: NGS consensus sequence 2 (P5R1SP) 118: DNA size is longer than 1000 bases 214: TS-FW primer (5'-end phosphorylation modification, black circle indicates phosphate group) 215: Amplification sequence - RV primer (5'-end phosphorylation modification, black circle indicates phosphate group) 216: C1-FW primer (uses the 5' sequence of the C region of the TCR (BCR) gene (mRNA)) 217: C1-RV primer (using the 3' sequence of the C region of the TCR (BCR) gene (mRNA)) 218: NGS consensus sequence 1 (P7R2SP) 219: NGS consensus sequence 2 (P5R1SP) 220: CT barcode 221: DNA size is shorter than 1000 bases 801: VFAC (a chip with 100 micro-reaction chambers (805) arranged in an array) 802: Through-hole at the top of the micro-reaction chamber 803: Cell 804: Packed beads (RT probe immobilized on the surface) 805: Single Cell and Microreactor 806: Liquid discharge part (porous material) 807: Downward pump suction 808: Lysed cell membrane 809: Dissolved nuclear membrane 810: mRNA

Claims

1. A method for simultaneously preparing a library for repertoire analysis of T cell receptors (TCRs) or B cell receptors (BCRs) and a library for gene expression analysis, comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) isolating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, and amplifying at least a portion of the target gene by PCR using a primer set utilizing the amplification sequence and a sequence specific to the target gene; A method comprising:

2. A method for simultaneous analysis of T cell receptor (TCR) or B cell receptor (BCR) repertoire analysis and gene expression analysis, comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) analyzing the sequence amplified by PCR in (6); (8) separating a portion of the amplification product obtained in the PCR amplification step (4) for gene expression analysis, and amplifying at least a portion of the target gene by PCR using a primer set utilizing the amplification sequence and a sequence specific to the target gene; (9) analyzing the sequence amplified by PCR in (8); A method comprising:

3. A method for preparing a library for repertoire analysis of T cell receptors (TCRs) or B cell receptors (BCRs), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; A method comprising:

4. A method for analyzing a repertoire of a T cell receptor (TCR) or a B cell receptor (BCR), comprising: (1) capturing mRNA eluted from cells using an RT probe immobilized on a solid surface, the RT probe including, in order from the 5' end, an amplification sequence, a barcode sequence, and an oligo(dT) sequence; (2) synthesizing a first cDNA using the captured mRNA as a template in a reverse transcription reaction solution containing a reverse transcriptase having a template switching function and a template switching oligo (TSO) to generate a first cDNA having a known sequence (TS) added to the 3' end; (3) removing excess RT probes that did not contribute to the synthesis of the first cDNA from the RT probes immobilized on the solid surface; (4) performing PCR amplification using the first cDNA as a template and a primer set utilizing the amplification sequence present at the 5' end of the first cDNA and the known sequence (TS) at the 3' end; (5) forming circular DNA from the amplification product of the PCR amplification step using ligase; (6) amplifying by PCR a region containing the barcode sequence and the variable region (VDJ) of TCR or BCR using the circular DNA as a template and a primer set in which both forward (FW) and reverse (RV) primers are designed using the sequence of the C region of TCR or BCR; (7) analyzing the sequence amplified by PCR in (6); A method comprising:

5. 5. The method of claim 1, wherein the barcode sequence comprises a cell identification barcode and a molecular identification barcode (UMI), and the cell is a single cell.

6. 5. The method of claim 1, wherein the barcode sequence comprises a molecular identification barcode (UMI) and the cell is a plurality of cells.

7. The method according to any one of claims 1 to 4, wherein in the PCR amplification step (6), a first primer set for TRA (or TRG) and a second primer set for TRB (or TRD) for TCR repertoire analysis are used as primer sets.

8. The method according to any one of claims 1 to 4, wherein in the PCR amplification step (6), five types of first primer sets for IgA / IgD / IgE / IgG / IgM for BCR repertoire analysis and two types of second primer sets for IgL / IgK for BCR-light chain analysis are used as primer sets.

9. The method according to any one of claims 1 to 4, wherein in the PCR amplification step (6), a primer set is used that is designed to be positioned near the 5' end and near the 3' end of the C region sequence so that the region to be PCR amplified is less than 1,000 bases.

10. The method according to any one of claims 1 to 4, wherein in the step (4) of carrying out PCR amplification, a primer set whose 5'-end is phosphorylated is used as the primer set.

11. The method according to any one of claims 1 to 4, wherein the step (3) of removing excess RT probes comprises decomposing the excess RT probes with Exonuclease I.

12. The method of claim 1 or 2, wherein the target gene comprises multiple genes.

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