Immune repertoire sequencing method

Abstract

Disclosed in the present invention is an immune repertoire sequencing method, comprising the following steps: 1) constructing a cDNA library of a sample, cDNA in the cDNA library being linked with position information of the cDNA on the sample; 2) carrying out targeted enrichment of cDNA of a TCR gene and / or a BCR gene in the cDNA library to obtain an immune repertoire of the sample; and 3) sequencing the immune repertoire. According to the method of the present invention, targeted enrichment and high-throughput sequencing of a TCR sequence and / or a BCR sequence are innovatively achieved, and at the same time, gene expression information and spatial position information of each cell in the space are detected.

Description

A sequencing method for immune repertoire Technical Field

[0001] The present invention belongs to the technical field of immune repertoire sequencing, and specifically relates to a method for sequencing an immune repertoire. Background Art

[0002] T and B lymphocytes selectively recognize antigens through alternative splicing of cell membrane surface receptors (TCR) and / or BCR genes, thereby achieving an acquired immune response. Immunoglobulin genes in B lymphocytes include IGH, IGK, and IGL, while T cell receptor genes in T lymphocytes include TRA and TRB. These genes are composed of a series of highly variable DNA regions, including variable (V), diversity (D), joining (J), and conserved constant (C) regions. Alternative splicing of these DNA regions can produce a variety of immune cell receptors.

[0003] B lymphocytes express B cell receptors (BCRs), which include multiple subtypes such as IgD, IgM, IgA, IgG, and IgE. B lymphocytes can produce antibodies that target specific antigens, and different B cells can produce different antibody clones. Antibodies are composed of two heavy chains (H chains) and two light chains (L chains). The heavy chain is encoded by the IGH gene and consists of a variable region and five conserved regions. The variable region includes the IGHV, IGHD, and IGHJ gene segments of the H chain, while the conserved regions include the gene segments of the five BCR subtypes, IGHG, IGHA, IGHM, IGHD, and IGHE. The light chain is encoded by the IGK and IGL genes and consists of a variable region and a conserved region. The variable regions include IGKV and IGKJ for the kappa chain and IGLV and IGLJ for the lambda chain. The highly variable region is called the Complementarity Determining Region (CDR), which includes CDR1, CDR2, and CDR3, and is separated by four structural regions (Framework Region, FR) FR1, FR2, FR3, and FR4.

[0004] T lymphocytes express cell-specific T cell receptors (TCRs) on their cell membranes, which play a crucial role in antigen presentation and recognition. When the TCR binds to a Major Histocompatibility Complex (MHC) molecule bound to an antigenic peptide, the T lymphocyte is activated, triggering a series of immune responses. The TCR is a heterodimeric protein composed of two highly variable polypeptide chains: the TCRα chain and the TCRβ chain. In humans, the TCRα chain is encoded by the TRA gene, and the TCRβ chain is encoded by the TRB gene. Both polypeptide chains include two extracellular domains: a variable region (V region) and a conserved region (C region). The variable region can bind to the antigenic peptide. The variable region of the TCR gene contains three highly variable CDRs, of which CDR3 is the primary binding site for recognizing the antigenic peptide. The conserved region of the TRA gene is called TRAC, and the conserved region of the TRB gene has two gene segments, called TRBC1 and TRBC2. The DNA sequences of the two segments are mostly the same, but there are also a few differences in the sequences.

[0005] The sum of the immunogenicity of all functionally diverse T and B cells in a given local space within an individual at a given time is called the local immune repertoire. High-throughput sequencing of the immune repertoire can greatly broaden our understanding of the human immune system and immune responses, and has broad application prospects.

[0006] Determining the sequence of TCR / BCR variable regions and, in turn, the antigenic specificity of TCR / BCR cell clones is a crucial area of ​​immunology research. Methods for specifically probing the local tissue immune repertoire are developing rapidly. However, given the length and variability of TCR / BCR gene fragments, developing an efficient and accurate method for determining their full-length sequences remains challenging. Currently, second-generation high-throughput sequencing methods typically only capture sequences adjacent to the UMI and barcode, limiting single-pass DNA sequencing read lengths to less than 500 bp. Conventional mRNA capture techniques utilize poly T probes that complement the poly A sequence of the mRNA and, through reverse transcription, position the UMI and barcode at the 3' end of the mRNA. However, for immune repertoire sequencing, the variable region at the 5' end of the mRNA is of particular interest to researchers, and this region is difficult to sequence using conventional sequencing technologies.

[0007] In summary, there is an urgent need in this field for an immune repertoire sequencing method that can measure spatiotemporal information.

[0008] Summary of the Invention

[0009] As mentioned above, there is an urgent need in this field for an immune repertoire sequencing method that can measure spatiotemporal information.

[0010] Based on existing spatiotemporal transcriptomics technology, the inventors innovatively achieved targeted enrichment and high-throughput sequencing of TCR and / or BCR sequences, simultaneously detecting gene expression information and spatial location information of each cell in space. This enabled the sequencing of the immune repertoire containing spatiotemporal information.

[0011] In a first aspect, the present invention provides a method for sequencing an immune repertoire, the method comprising the following steps:

[0012] 1) constructing a cDNA library of the sample, wherein the cDNA in the cDNA library is connected to its position information on the sample;

[0013] 2) Targeted enrichment of TCR and / or BCR gene cDNAs in the cDNA library to obtain the immune repertoire of the sample;

[0014] 3) Sequencing the immune repertoire.

[0015] In a second aspect, the present invention provides primers for an immune repertoire sequencing method, wherein the primers achieve the targeted enrichment of cDNA of TCR and / or BCR genes in the method described in the first aspect, and the primers are shown in SEQ ID NO: 337-SEQ ID NO: 347.

[0016] In a third aspect, the present invention provides a kit for performing immune repertoire sequencing, wherein the kit utilizes the method described in the first aspect to perform immune repertoire sequencing.

[0017] In the fourth aspect, the present invention provides the use of the method described in the first aspect, the primers described in the second aspect or the kit described in the third aspect, and the use includes but is not limited to: identifying the location of immune lymphocytes in tissues, the spatiotemporal pairing of TCR α chain and β chain, identifying cellular immunogenicity and multiple cell membrane immune receptor CDR3 gene sequences.

[0018] The method of the present invention innovatively achieves targeted enrichment and high-throughput sequencing of TCR and / or BCR sequences, while simultaneously detecting the gene expression information and spatial location information of each cell in space; and the method of the present invention has the advantages of high sequencing sensitivity and strong versatility, which is conducive to commercial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the specific embodiments of the present invention, the drawings in the specific embodiments are briefly introduced below.

[0020] FIG1 shows a schematic diagram of capturing TRA, TRB, and IGH gene transcripts in frozen tissue sections using a Stereo-seq chip.

[0021] FIG2 is a schematic diagram showing the process of enriching TCR / BCR gene cDNA using V region targeting primers, amplifying the enriched cDNA, and adding sequencing barcodes.

[0022] FIG3 shows a schematic diagram of circularizing single-stranded DNA using the circularization primer Splint Oligo.

[0023] FIG4 shows a schematic diagram of circularizing double-stranded DNA using the Gibson recombination method.

[0024] FIG5 shows a schematic diagram of a process for further enriching the CDR3 region of TCR and / or BCR genes using TCR and / or BCR Target CirDNA Primer as a primer.

[0025] FIG6 shows a schematic diagram of the immune repertoire library DNB preparation and sequencing scheme.

[0026] 7A and B show the sequencing results of the non-enriched sample and the TRB-targeted enriched sample in Example 1, respectively.

[0027] Figure 8 shows the sequencing results for the unenriched samples (control group) and the gene-enriched samples (experimental group) in Examples 2 and 3. The control group shows the sequencing results of the TRBC gene detected by the unenriched spatiotemporal transcriptome, while the experimental group shows the sequencing results of the V regions enriched for the human TRA, TRB, and IGH genes, respectively. The scale in the figure represents the number of UMIs measured in bin 200.

[0028] Figure 9 shows the sequencing results of the targeted enrichment of the TRA gene in Example 2. Panel A shows the measured CDR3 amino acid sequence length; Panel B shows the types and sequencing numbers of the measured TRA gene V and J regions; Panel C shows the spatial distribution of the most frequently measured CDR3 clonotype (CASSFYDRGEQYF).

[0029] Figure 10 shows the sequencing results of the targeted enrichment of the TRB gene in Example 2. Panel A shows the measured CDR3 amino acid sequence length; Panel B shows the types and sequencing counts of the TRB gene V and J regions; Panel C shows the spatial distribution of the most frequently detected CDR3 clonotype (CAIGLAGGDEQYF).

[0030] Figure 11 shows the sequencing results of the targeted enrichment of the IGH gene in Example 3. Panel A shows the measured CDR3 amino acid sequence length; Panel B shows the types and sequence counts of the measured IGH gene V and J regions; Panel C shows the spatial distribution of the most frequently measured CDR3 clonotype (CAINRYTRGAEFW).

[0031] Figure 12 shows the sequencing results of the targeted enrichment mouse Tcra gene in Example 4. Panel A shows the measured CDR3 amino acid sequence length; Panel B shows the types and sequencing numbers of the measured Tcra gene V and J regions; Panel C shows the spatial distribution of the most measured CDR3 clonotype (YLCAAGARSNNRIFF).

[0032] Figure 13 shows the sequencing results of the targeted enrichment of the mouse Tcrb gene in Example 4. Panel A shows the measured CDR3 amino acid sequence length; Panel B shows the types and sequencing numbers of the measured Tcrb gene V and J regions; Panel C shows the spatial distribution of the most frequently measured CDR3 clonotype (CASSFYDRGEQYF). DETAILED DESCRIPTION

[0033] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is merely illustrative of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. Furthermore, those skilled in the art will appreciate that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0035] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the subject matter.

[0036] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can generally be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed-ended expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0037] For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood as being modified in all cases by the term "about", unless otherwise indicated. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0038] The term "sample" refers to fresh tissue after sampling, which is prepared as a frozen sample, a paraffin sample, or a slice as required, but is not limited thereto.

[0039] The term "primer" refers to a naturally occurring oligonucleotide (e.g., a restriction fragment) or a synthetically produced oligonucleotide that is capable of serving as a point of initiation for the synthesis of a primer extension product that is complementary to a nucleic acid strand (template or target sequence) when placed under appropriate conditions (e.g., buffer, salts, temperature, and pH) and in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., a DNA-dependent or RNA-dependent polymerase).

[0040] The term "targeted enrichment" refers to the capture and / or amplification of target DNA. For example, the enrichment process can increase the cDNA of TCR and / or BCR genes by several fold compared to the cDNA of non-TCR and / or BCR genes.

[0041] The term "immune repertoire" refers to the sum of all functionally diverse B and T cells within the local tissues of an individual at any given time.

[0042] The term "Gibson assembly" refers to a molecular cloning method for assembling multiple linear DNA fragments or inserting a fragment of interest into a vector of choice. Compared to other cloning methods that utilize shorter repeat regions between fragments, the Gibson assembly method utilizes long repeat regions between fragments to more specifically ensure the order of ligation.

[0043] The term "uracil-specific excision reagent" refers to any material that functions in excising a uracil residue converted from a cytosine residue by a cytosine deaminase and / or inducing DNA cleavage at the site of uracil excision.

[0044] As mentioned above, the present invention aims to provide a method for sequencing immune repertoires containing spatiotemporal information.

[0045] Therefore, in a first aspect, the present invention provides a method for sequencing an immune repertoire, the method comprising the following steps:

[0046] 1) constructing a cDNA library of the sample, wherein the cDNA in the cDNA library is connected to its position information on the sample;

[0047] 2) Targeted enrichment of TCR and / or BCR gene cDNAs in the cDNA library to obtain the immune repertoire of the sample;

[0048] 3) Sequencing the immune repertoire.

[0049] Each step of the method is described in detail below.

[0050] The method used in step 1) includes but is not limited to Stereo-seq. Figure 1 shows a schematic diagram of using a Stereo-seq chip to capture TRA gene, TRB gene and IGH gene transcripts in frozen tissue sections. The Stereo-seq chip can capture mRNA in frozen tissue sections, generate cDNA through reverse transcription, and release cDNA with spatial position information for PCR amplification using universal primers. As shown in Figure 1, 154M is the fixed sequence and universal primer sequence on the Stereo-seq chip probe, which is used for PCR amplification. CID (Coordinate ID) is a DNA sequence that encodes spatial position information. The CID sequence length of the standard Stereo-seq library is 25bp. The CID can be used to restore the spatial position of the subsequently captured mRNA. UMI is a unique molecular index. The Poly T sequence on the chip is used to capture the Poly A of the mRNA. After processing frozen tissue sections plated on the chip, mRNA molecules within them bind to the poly T sequence and are reverse-transcribed using MMLV reverse transcriptase to generate cDNA. Through template displacement, a TSO primer containing the 154M adapter sequence for PCR amplification is added to the 3' end of the cDNA. The captured TRA and TRB gene mRNAs are approximately 1 kb in length, and the captured IGH gene mRNA is approximately 1.5 kb in length.

[0051] Step 2) specifically includes: capturing and amplifying the cDNA of the TCR and / or BCR genes in the cDNA library, circularizing the captured and amplified cDNA to obtain circular single-stranded DNA or circular double-stranded DNA, purifying the circular single-stranded DNA or circular double-stranded DNA, and further amplifying the circular single-stranded DNA or circular double-stranded DNA to obtain the immune repertoire of the sample.

[0052] In step 2), the cDNA of described targeted enrichment TCR and / or BCR gene is realized by using targeting primer, and described targeting primer amplifies the variable region of TCR and / or BCR gene in a PCR reaction.Described targeting primer can target CDR1, CDR2 or CDR3 region upstream 10bp-30bp or 50bp-150bp of TCR and / or BCR gene variable region.For the variable region of different sequences, corresponding targeting primer can be designed, and targeting primer sequence is optimized by multiple primer design software so that it can be used for a PCR reaction simultaneously.For example, when designing the targeting primer of targeting TCR variable region, design targeting homology but the targeting degenerate primer of different types of TRAV, TRBV can cover the variable (V) region of most TRA, TRB genes, including CDR1, CDR2, CDR3 region.In one embodiment, the targeting primer of targeting human TRA gene variable (V) district can be 43 primers as shown in Table 1, and these primers are in conjunction with the upstream of CDR3. In one embodiment, the targeting primers targeting the variable (V) region of the human TRB gene can be 26 primers as shown in Table 2, which bind to the upstream of CDR3. In one embodiment, the targeting primers targeting the variable (V) region of the human IGH gene can be 12 primers as shown in Table 3, which bind to the upstream of CDR3. In one embodiment, the targeting primers targeting the variable (V) region of the mouse Tcra gene can be 20 primers as shown in Table 4, which bind to the upstream of CDR3. In one embodiment, the targeting primers targeting the variable (V) region of the mouse Tcrb gene can be 22 primers as shown in Table 5, which bind to the upstream of CDR3. In one embodiment, the targeting primers targeting the variable (V) region of the mouse Igh gene can be 16 primers as shown in Table 6, which bind to the upstream of CDR3. It should be understood that the targeting primers for targeting and enriching the cDNA of the TCR and / or BCR genes in the present invention are not limited to the primers described in Tables 1-6, and this purpose can also be achieved using other suitable primers. In one embodiment, the targeting primer (excluding the sequencing adapter) is 18 bp to 30 bp in length. In one embodiment, the final concentration of the targeting primer combination is 0.2 μM to 0.5 μM. In one embodiment, the annealing temperature of the targeting primer is 50° C. to 70° C.

[0053] Table 1: Primers targeting CDR3 in the V region of the human TRA gene

[0054] Table 2: Primers targeting CDR3 in the V region of the human TRB gene

[0055] Table 3: Primers targeting human IGH gene V region CDR3

[0056] Table 4: Primers targeting mouse Tcra gene V region CDR3

[0057] Table 5: Primers targeting mouse Tcrb gene V region CDR3

[0058] Table 6: Primers targeting mouse Igh gene V region CDR3

[0059] Figure 2 shows a schematic diagram of the cDNA structure containing TCR / BCR genes captured using the Stereo-seq chip and a schematic diagram of the PCR amplification enrichment process. The targeted primers used to enrich TCR / BCR cDNA include primer sequences that specifically bind to the TCR / BCR V region framework structure, and a linker primer sequence (V3-181 sequence) for PCR amplification. After amplification with the targeted primers, a DNA product containing the TCR / BCR CDR3 region can be obtained. The captured cDNA containing TRA and TRB genes is 1kb in length, and the cDNA of the IGH gene is 1.5kb in length. After PCR amplification by the targeted primers, the DNA transcript is about 200bp-400bp. Afterwards, a universal Barcode sequence can be added to distinguish sequencing samples in high-throughput sequencing.

[0060] In one embodiment, in step 2), the method for obtaining circular single-stranded DNA comprises: denaturing the cDNA molecule at high temperature, applying primers such as Splint Oligo primers to both ends of the cDNA molecule, and ligating the ends using a DNA ligase such as T4 DNA ligase to obtain circular single-stranded DNA. Figure 3 shows a schematic diagram of circularizing single-stranded DNA using the circularization primer Splint Oligo. Splint Oligo primer PGMR-BP2: One end of Splint Oligo is complementary to the barcode primer adapter (BP2) and the other end is complementary to the universal primer (PGMR), thereby achieving circularization of the single-stranded DNA.

[0061] In one embodiment, in step 2), the method for obtaining circular double-stranded DNA is to design homology arm sequences required for Gibson recombination, add the homology arm sequences to the amplification primers of the cDNA, and obtain circular double-stranded DNA via the Gibson recombination method. As shown in Figure 4, homology sequences are added to the 5' ends of the targeting primer and the universal primer for the amplified gene, respectively. Using the Gibson recombination method, the linear DNA amplified by PCR can be circularized using V3-181 to form circular double-stranded DNA. The length of the circular DNA is approximately 400 bp–2 kb.

[0062] In one embodiment, in step 2), the method for obtaining circular double-stranded DNA is: connecting a linker primer sequence containing uracil to the targeting primer, applying a uracil-specific excision reagent (such as USER enzyme, NEB) to cut the cDNA of the TCR and / or BCR gene to generate sticky ends to achieve double-stranded circularization.

[0063] In one embodiment, in step 2), the method for purifying circular single-stranded DNA or circular double-stranded DNA is: after the cyclization reaction is completed, an enzyme with 3'-5' exonuclease activity (such as Exo I and Exo III DNA exonucleases) is added to the cyclization system to digest and decompose the uncircularized DNA molecules, and then the circular DNA is purified using magnetic beads.

[0064] In one embodiment, in step 2), the method for further amplifying the circular single-stranded DNA or circular double-stranded DNA is to perform PCR amplification using primers targeting the conserved regions of the TCR and / or BCR genes and the circular DNA of the TCR and / or BCR genes as a template. A schematic diagram of this method is shown in Figure 5. Amplification primers targeting the conserved regions of the human TRA, TRB, and IGH genes and the mouse Tcra, Tcrb, and Igh genes are described in detail below, and some of the primers are shown in Table 7.

[0065] In one embodiment, as shown in Figure 5, primers targeting the conserved regions of the human TRB gene, termed TRB Target CirDNA Primers, are designed. The conserved regions of the TRB gene are divided into TRBC1 and TRBC2. The forward primer of the TRB Target CirDNA Primer binds to the homologous sequence region of TRBC1 and TRBC2 and is located at the 3' end of the CDS region of the gene, 10-100 bp from the stop codon. The reverse primer of the TRB Target CirDNA Primer binds to the homologous sequence region of TRBC1 and TRBC2 and is located at the 5' end of the CDS region of the gene, 10-100 bp from the CDS start site of the conserved region of the TRB gene. Using the DNA product of the targeted enriched TRB gene transcript as a template and the TRB Target CirDNA Primer as primers, PCR can be used to amplify and enrich DNA molecules of the variable region of the TRB gene containing sequences with temporal and spatial localization information. The PCR product is approximately 200–600 bp in length and can be purified and isolated using magnetic beads.

[0066] In one embodiment, as shown in FIG5 , primers targeting the conserved region of the mouse Tcrb gene, called mTcrb Target CirDNA Primer, are designed. The above method can be used, with the DNA product targeting and enriching the mouse Tcrb gene transcript as a template and the mTcrb Target CirDNA Primer as a primer, to PCR amplify and enrich the Tcrb gene variable region DNA molecules containing the spatiotemporal localization information sequence. The PCR product is approximately 200 bp–600 bp in length, and the DNA product can be purified and separated using magnetic beads.

[0067] In one embodiment, as shown in Figure 5, primers targeting the conserved region of the human TRA gene, termed TRA Target CirDNA Primer, are designed. The forward primer of the TRA Target CirDNA Primer binds to the 3' end of the CDS region of the conserved region TRAC of the TRA gene, 10-100 bp from the stop codon; the reverse primer of the TRA Target CirDNA Primer is located at the 5' end of the CDS region of the gene, 10-100 bp from the start site of the CDS of the conserved region of the TRA gene. Using the DNA product of the targeted enriched TRA gene transcript as a template and the TRA Target CirDNA Primer as primers, PCR can be used to amplify and enrich DNA molecules of the variable region of the TRA gene containing sequences with temporal and spatial localization information. The PCR product is approximately 200–600 bp in length and can be purified and isolated using magnetic beads.

[0068] In one embodiment, as shown in FIG5 , primers targeting the conserved region of the mouse Tcra gene, called mTcra Target CirDNA Primer, are designed. The above method can be used, using the DNA product targeted to enrich the mouse Tcra gene transcript as a template and the mTcra Target CirDNA Primer as a primer, to PCR amplify and enrich the Tcra gene variable region DNA molecules containing the spatiotemporal localization information sequence. The PCR product is approximately 200 bp–600 bp in length, and the DNA product can be purified and separated using magnetic beads.

[0069] In one embodiment, as shown in Figure 5, primers targeting the conserved regions of the human IGH gene are designed, referred to as the IGH Target CirDNA Primer Mix. The human IGH gene has five conserved regions, namely IGHG, IGHA, IGHM, IGHD, and IGHE. The IGH Target CirDNA Primer Mix includes amplification primers targeting each IGH genotype, for a total of 10 primers. The Forward Primer of the IGH Target CirDNA Primer binds to the 3' end of the CDS region of the conserved region of the IGH gene, 10-100 bp from the stop codon; the Reverse Primer of the IGH Target CirDNA Primer is located at the 5' end of the CDS region of the gene, 10-100 bp from the start site of the CDS of the conserved region of the IGH gene. Using the DNA product of targeted enrichment of IGH gene transcripts as a template and the IGH Target CirDNA Primer Mix as primers, PCR can be used to amplify and enrich DNA molecules in the variable region of the IGH gene containing temporal and spatial localization information sequences. The PCR product is approximately 200bp–600bp in length and can be purified and separated using magnetic beads.

[0070] In one embodiment, as shown in FIG5 , primers targeting the conserved region of the mouse Igh gene are designed, referred to as the mIgh Target CirDNA Primer Mix. The mouse Igh gene has five conserved region types, namely Ighg, Igha, Ighm, Ighd, and Ighe. The mIgh Target CirDNA Primer Mix includes amplification primers targeting each Igh genotype, for a total of 10 primers. The Forward Primer of the mIgh Target CirDNA Primer binds to the 3' end of the CDS region of the conserved region of the Igh gene, 10-100 bp from the stop codon; the Reverse Primer of the mIgh Target CirDNA Primer is located at the 5' end of the CDS region of the gene, 10-100 bp from the start site of the CDS of the conserved region of the Igh gene. Using the DNA product targeted to enrich mouse Igh gene transcripts as a template and the mIgh Target CirDNA Primer Mix as primers, PCR can be used to amplify and enrich DNA molecules in the variable region of the Igh gene containing temporal and spatial localization information sequences. The PCR product is approximately 200bp–600bp in length and can be purified and separated using magnetic beads.

[0071] Table 7: Amplification primers targeting the conserved regions of human TRA, TRB, IGH genes and mouse Tcra, Tcrb, Igh genes

[0072] The Target CirDNA Primer is designed with primer adapter sequences compatible with the BGI Universal Sequencing Kit. The MGISEQ-2000RS High-Throughput Sequencing Reagent Set (Cat. No. 1000013858) enables one-step DNA nanoball (DNB) preparation for high-throughput sequencing. Figure 6 shows a schematic diagram of the immune panel library DNB preparation and sequencing protocol.

[0073] In one embodiment, in step 3), the sequencing method includes but is not limited to: using BGI second-generation sequencing technology to prepare DNBs and sequence the immune repertoire. It should be understood that other suitable sequencing methods can also achieve the purpose of the present invention.

[0074] In a second aspect, the present invention provides primers for an immune repertoire sequencing method, wherein the primers achieve the targeted enrichment of cDNA of TCR and / or BCR genes in the method described in the first aspect, and the primers are shown in SEQ ID NO: 337-SEQ ID NO: 347 (Table 8).

[0075] Table 8: Primers used for immune repertoire sequencing method

[0076] In a third aspect, the present invention provides a kit for performing immune repertoire sequencing, wherein the kit utilizes the method described in the first aspect to perform immune repertoire sequencing.

[0077] In the fourth aspect, the present invention provides the use of the method described in the first aspect, the primers described in the second aspect or the kit described in the third aspect, and the use includes but is not limited to: identifying the location of immune lymphocytes in tissues, the spatiotemporal pairing of TCR α chain and β chain, identifying cellular immunogenicity and multiple cell membrane immune receptor CDR3 gene sequences.

[0078] Example

[0079] It should be noted that the terms used in the description of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The above summary of the invention and the detailed description below are intended only to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the appended claims.

[0080] Example 1

[0081] TRB cDNA molecules were enriched from a human tonsil spatiotemporal transcriptome cDNA library using a primer combination targeting the variable region of the TRB gene (primer sequences are detailed in Table 2). The spatiotemporal transcriptome cDNA library was prepared using the standard BGI STOmics protocol. Using 100 ng of spatiotemporal transcriptome cDNA with 154M sequences at both ends as template, 1 μmol of cDNA Reverse Primer, 4 μmol of the TRBV targeted primer combination, and 50 μL of PCR Amplification Mix were added, and the volume was filled to 100 μL with deionized water. PCR conditions were 98°C for 20 s, 58°C for 20 s, and 72°C for 45 s, with 30 cycles. PCR products were purified using 0.6X VATHS Beads (Vazyme), eluted with 42 μL of deionized water, and DNA concentration was determined using a Qubit assay.

[0082] To test whether TRB genes were enriched, a spatiotemporal sequencing cDNA library was prepared using the sequencing library construction kit included in the STOmics kit. The following steps were performed: 40 ng of TRB targeted amplification cDNA product was added to 4 μL of TMB reagent and 1 μL of a ten-fold dilution of TME reagent, which was then made up to 20 μL with deionized water. The reaction was then placed in a PCR instrument and incubated at 55°C for 10 minutes. 5 μL of STOP buffer was added at room temperature, mixed thoroughly, and the reaction was terminated by incubation at room temperature for 5 minutes. Next, 25 μL of PCR Barcode Primer Mix and 50 μL of PCR Amplification Mix were added to the reaction system, resulting in a total volume of 100 μL. PCR conditions were 98°C for 20 seconds, 58°C for 20 seconds, and 72°C for 30 seconds, for 13 cycles. The PCR product was purified and recovered using 0.55X-0.15X VATHS beads (Vazyme), eluted with 22 μL of deionized water, and DNA concentration was determined using a Qubit assay.

[0083] Take 40 ng of the sorted cDNA product and use the MGISEQ-2000RS High-Throughput Sequencing Reagent Set (Cat. No. 1000013858) for One-Step DNB preparation. The prepared DNB is loaded onto the sequencing slide and sequenced.

[0084] The results are shown in Figure 7. The number of unique reads for the unenriched sample was 24.14M, and the number of reads for TRBC was 7.9k. The number of unique reads for the TRB-targeted enriched sample was 23.72M, and the number of reads for TRBC was 1.2M. Compared to the whole transcriptome, the number of reads for the TRBC gene was significantly higher in the TRB-targeted enriched sample, with the degree of enrichment increasing by over 100-fold.

[0085] Example 2

[0086] PCR amplification was performed using a combination of targeting primers targeting the variable regions of the TRA and TRB genes and a 5'-phosphorylated adapter primer to obtain human tonsil cDNA fragments of interest for the TRA and TRB genes, which served as substrates for the circularization reaction. Splint Oligo primers were then designed to complement the targeting primer adapters and universal primers. 100 ng–1 μg of the amplified cDNA product was added to 12 μL of Splint Buffer and made up to 60 μL with double-distilled water. The reaction was placed in a PCR instrument, denatured at 95°C for 3 minutes, then annealed at 40°C for 3 minutes, removed, and placed on ice. At this point, one end of the Splint Oligo primer was complementary to the targeting primer adapter sequence, and the other end was complementary to the universal primer sequence of the same cDNA molecule, forming a circular structure. 1 μL of T4 DNA ligase was then added for ligation, and the reaction was incubated at 37°C for 30 minutes. The single-stranded DNA complementary to the Splint Oligo primers was ligated end-to-end to form circular DNA. The circular DNA ranged in size from approximately 400 bp to 2 kb. Afterwards, DNA was purified and recovered using 100 μL of VATHS Beads, eluted with 42 μL of double-distilled water, and the DNA concentration was determined using Qubit.

[0087] After circularization of the targeted enriched TRA or TRB gene molecules, amplification was performed using TCP primers. Using the circularized, targeted enriched DNA product as a template, 2 μmol of TRA / TRB TCP Primer Mix and 50 μL of PCR Amplification Mix were added, and the volume was made up to 100 μL with redistilled water. PCR conditions were 98°C for 20 seconds, 55°C for 20 seconds, and 72°C for 45 seconds, with 30 cycles. PCR products were purified and recovered using 0.8X VATHS Beads (Vazyme), eluted with 42 μL of redistilled water, and DNA concentration was determined using a Qubit assay. 200 ng of PCR product was used to prepare DNA bonds (DNBs) and sequenced according to standard procedures. The sequencing results are shown in Figures 8-10, demonstrating the ability to determine multiple, efficiently enriched TCR CDR3 gene sequences containing spatiotemporal information.

[0088] After bioinformatics analysis, the results are shown in Figures 9 and 10. In the tonsil section of this person, a total of 5.3×10 TRA CDR3 types can be identified. 5 There are 1.99×10 TRB CDR3 types in total. 6The amino acid sequence lengths are normal. Sequencing results cover a wide range of V and J region sequences, demonstrating minimal sequence bias during targeted amplification and library construction. Based on spatial coordinates, CDR3 sequences can be mapped to spatial locations and spatial maps can be created. This spatial location information can also be used to localize TRA and TRB CDR3 sequences to different tissue locations, enabling spatially informed immune repertoire sequencing and paired analysis.

[0089] Example 3

[0090] PCR amplification was performed using a combination of targeting primers targeting the variable region of the IGH gene and a 5'-phosphorylated adapter primer to obtain a cDNA fragment of the human tonsil IGH gene as the substrate for the circularization reaction. 100 ng–1 μg of the amplified cDNA product targeting the IGH gene was added to 6 μL of 10X T4 Ligase Buffer and 6 μL of 10 μM Splint Oligo primer, and the mixture was made up to 60 μL with double-distilled water. The reaction mixture was placed in a PCR instrument, denatured at 95°C for 3 minutes, then annealed at 40°C for 3 minutes, removed, and placed on ice. At this point, one end of the Splint Oligo primer was complementary to the adapter sequence of the targeting primer, and the other end of the Splint Oligo primer was complementary to the universal primer sequence of the same cDNA molecule, forming a circular structure. 1 μL of T4 DNA ligase was then added for ligation, and the reaction was incubated at 37°C for 30 minutes. The single-stranded DNA complementary to the Splint Oligo primer was ligated end-to-end to form a circular DNA. The circular DNA ranged in size from approximately 400 bp to 2 kb. Afterwards, DNA was purified and recovered using 100 μL of VATHS Beads, eluted with 42 μL of double-distilled water, and the DNA concentration was determined using Qubit.

[0091] After circularization of the targeted enriched IGH molecules, amplification was performed using TCP primers. Using the circularized targeted enriched IGH DNA product as template, 4 μmol of IGH TCP Primer Mix and 50 μL of PCR Amplification Mix were added, and the volume was made up to 100 μL with redistilled water. PCR conditions were 98°C for 20 s, 55°C for 20 s, and 72°C for 60 s, with 30 cycles. The PCR product was purified and recovered using 0.8X VATHS Beads (Vazyme), eluted with 42 μL of redistilled water, and the DNA concentration was determined using a Qubit assay. DNA DNA was prepared from 200 ng of PCR product and sequenced according to standard protocols. The sequencing results are shown in Figures 8 and 11, demonstrating the ability to efficiently and efficiently enrich multiple IGH CDR3 gene sequences containing spatiotemporal information.

[0092] After bioinformatics analysis, the results are shown in Figure 11. In this person's tonsil section, a total of 2.0×10 IGH CDR3 types can be identified. 6 The amino acid sequence lengths were normal. Sequencing results cover a wide range of V and J region sequences, demonstrating minimal sequence bias during targeted amplification and library construction. Spatial coordinates can be used to map CDR3 sequences to spatial locations, enabling spatially informed immune repertoire sequencing.

[0093] Example 4

[0094] PCR amplification was performed using a combination of targeting primers targeting the variable regions of the mouse Tcra and Tcrb genes and a 5'-phosphorylated adapter primer to obtain cDNA fragments of the mouse Tcra and Tcrb genes as substrates for circularization. 100 ng–1 μg of thymic cDNA amplified from mouse Tcra and Tcrb, respectively, was added to 6 μL of 10X T4 Ligase Buffer and 6 μL of 10 μM Splint Oligo primers, and the mixture was made up to 60 μL with double-distilled water. The reaction mixture was placed in a PCR instrument, denatured at 95°C for 3 minutes, then annealed at 40°C for 3 minutes, removed, and placed on ice. At this point, one end of the Splint Oligo primer was complementary to the adapter sequence of the targeting primer, and the other end of the Splint Oligo primer was complementary to the universal primer sequence of the same cDNA molecule, forming a circular structure. 1 μL of T4 DNA ligase was then added, and the reaction was incubated at 37°C for 30 minutes. The single-stranded DNA complementary to the Splint Oligo primers was now ligated end-to-end, forming a circular DNA. The circular DNA is approximately 400 bp–2 kb in size. DNA was then purified and recovered using 100 μL of VATHS beads, eluted with 42 μL of double-distilled water, and the DNA concentration was determined using a Qubit assay.

[0095] After circularization of the targeted and enriched mouse Tcra and Tcrb molecules, amplification was performed using TCP primers. Using the circularized, targeted and enriched mouse Tcra and Tcrb DNA products as templates, 2 μmol of mTcra / mTcrb TCP Primer Mix and 50 μL of PCR Amplification Mix were added, and the volume was made up to 100 μL with distilled water. PCR conditions were 98°C for 20 s, 55°C for 20 s, and 72°C for 60 s, with 30 cycles. The PCR product was purified using 0.8X VATHS Beads (Vazyme), eluted with 42 μL of distilled water, and the DNA concentration was determined using a Qubit assay. DNA was prepared from 200 ng of PCR product and sequenced according to standard protocols.

[0096] After bioinformatics analysis, the results are shown in Figures 12 and 13. In the thymus sections of this mouse, a total of 4.7×10 Tcra CDR3 types can be identified. 5 There are 4.3×10 Tcrb CDR3 types in total. 5 The amino acid sequence lengths were normal. Sequencing results cover a wide range of V and J region sequences, demonstrating minimal sequence bias during targeted amplification and library construction. CDR3 sequences can be mapped to spatial locations based on spatial coordinates, allowing for the creation of a spatial map.

[0097] Currently, immune repertoire sequencing technologies based on spatiotemporal transcriptome technology have not been commercially applied. Hudson et al. reported that using primers targeting the human TRB gene, they targeted the TRB CDR3 region within spatiotemporal transcriptome cDNA prepared using the 10X Genomics Visium Spatial Gene Expression platform, and sequenced it, providing T cell clonotype information from tumor-infiltrating samples. In contrast, we utilized BGI's spatiotemporal transcriptome technology to design targeted primers targeting the V and C regions of TCR and / or BCR molecules, enabling secondary enrichment of CDR3 gene segments. This improved sequencing quality, significantly reduced sequencing load, and enabled paired analysis of TCR α and β chains. Engblom et al. reported that using a specialized TCR / BCR spatiotemporal targeted capture chip, they were able to obtain enriched TCR / BCR cDNA. Using PacBio real-time single-molecule DNA sequencing, they obtained full-length TCR / BCR sequences and, through targeted amplification, obtained TCR / BCR CDR3 sequences. In comparison, we use a universal spatiotemporal transcriptome capture chip that can capture human and mouse TCR and / or BCR molecules and perform targeted enrichment, obtain CDR3 sequence information of TCR and / or BCR, and have the potential for full-length sequencing.

[0098] The above is a detailed introduction to the sequencing method of the immune repertoire provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above examples is only used to help understand the method of the present invention and its core ideas. At the same time, for those skilled in the art, according to the ideas of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for sequencing an immune repertoire, characterized in that: The method comprises the following steps: 1) constructing a cDNA library of a sample, wherein the cDNA in the cDNA library is connected with its position information on the sample; 2) Targeted enrichment of cDNA of TCR and / or BCR genes in the cDNA library to obtain the immune repertoire of the sample; 3) Sequencing the immune repertoire.

2. The method according to claim 1, characterized in that: In step 2), the targeted enrichment of TCR and / or BCR gene cDNA specifically includes: capturing and amplifying the TCR and / or BCR gene cDNA in the cDNA library, circularizing the captured amplified cDNA to obtain circular single-stranded DNA or circular double-stranded DNA, purifying the circular single-stranded DNA or circular double-stranded DNA, and further amplifying the circular single-stranded DNA or circular double-stranded DNA.

3. The method according to claim 1 or 2, characterized in that: In step 2), the targeted enrichment of cDNA of TCR and / or BCR genes is achieved by using targeting primers, which amplify the variable regions of TCR and / or BCR genes in one PCR reaction.

4. The method according to claim 3, characterized in that The targeting primers are selected from at least one group of the following sequences: targeting primers targeting the human TRA gene V region SEQ ID NO:1-SEQ ID NO:86; targeting primers targeting the human TRB gene V region SEQ ID NO:87-SEQ ID NO:138; targeting primers targeting the human IGH gene V region SEQ ID NO:139-SEQ ID NO:162; targeting primers targeting the mouse Tcra gene V region SEQ ID NO:163-SEQ ID NO:202; targeting primers targeting the mouse Tcrb gene V region SEQ ID NO:203-SEQ ID NO:246; targeting primers targeting the mouse Igh gene V region SEQ ID NO:247-SEQ ID NO:

278.

5. The method according to claim 3 or 4, characterized in that: Without including a sequencing adapter, the targeting primer is 18 bp to 30 bp in length.

6. The method according to any one of claims 3 to 5, characterized in that: The final concentration of the targeting primer combination is 0.2 μM-0.5 μM.

7. The method according to any one of claims 3 to 6, characterized in that: The targeting primer annealing temperature is 50°C-70°C.

8. The method according to any one of claims 2 to 7, characterized in that: The method for obtaining circular single-stranded DNA is: denaturing the cDNA molecule at high temperature, using primers such as Splint Oligo primers to bind to both ends of the cDNA molecule, and connecting with DNA ligase such as T4 DNA ligase to obtain circular single-stranded DNA.

9. The method according to any one of claims 2 to 7, characterized in that: The method for obtaining circular double-stranded DNA is: designing homology arm sequences required for Gibson recombination, adding the homology arm sequences to the amplification primers of the cDNA, and obtaining circular double-stranded DNA by the Gibson recombination method.

10. The method according to any one of claims 2 to 7, characterized in that: The method for obtaining circular double-stranded DNA is: connecting a linker primer sequence containing uracil to the targeting primer, using a uracil-specific excision reagent to cut the cDNA of the TCR and / or BCR gene to generate sticky ends to achieve double-stranded circularization.

11. The method according to any one of claims 2 to 10, characterized in that: In step 2), the method for purifying circular single-stranded DNA or circular double-stranded DNA is: after the cyclization reaction is completed, enzymes with 3'-5' exonuclease activity are added to the cyclization system to digest and decompose the uncyclized DNA molecules, and then the circular DNA is purified using magnetic beads.

12. The method according to any one of claims 2 to 11, characterized in that: In step 2), the method for further amplifying the circular single-stranded DNA or circular double-stranded DNA is: using primers of the conserved region of the TCR and / or BCR genes, and taking the circular DNA of the TCR and / or BCR genes as a template, to perform PCR amplification; wherein the primers of the conserved region of the TCR and / or BCR genes include amplification primers targeting the conserved region of human TRA, TRB, IGH genes or mouse Tcra, Tcrb, Igh genes, and are selected from at least one primer among SEQ ID NO: 279-SEQ ID NO:

336.

13. The method according to any one of claims 1 to 12, characterized in that The techniques used in step 1) include but are not limited to: Stereo-seq.

14. The method according to any one of claims 1 to 13, characterized in that In step 3), the sequencing method includes but is not limited to: using BGI's second-generation sequencing technology to prepare DNBs and sequence the immune repertoire.

15. Primers for immune repertoire sequencing method, characterized in that, The primers achieve the targeted enrichment of cDNA of TCR and / or BCR genes in the method according to any one of claims 1 to 14, and the primers are shown in SEQ ID NO: 337-SEQ ID NO:

347.

16. A kit for performing immune repertoire sequencing, characterized in that: The kit uses the method described in any one of claims 1-14 to achieve immune repertoire sequencing.

17. Use of the method according to any one of claims 1 to 14, the primer according to claim 15 or the kit according to claim 16, characterized in that: The applications include but are not limited to: identifying the location of immune lymphocytes in tissues, the spatiotemporal pairing of TCR α chain and β chain, identifying cellular immunogenicity and various cell membrane immune receptor CDR3 gene sequences.

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