Space-time transcriptome specific enrichment method and application thereof

Abstract

The invention provides a space-time transcriptome specific enrichment method and application thereof.The method comprises the steps that firstly, a capture primer is adopted to enrich space-time transcriptome cDNA library products, then magnetic beads are adopted to conduct fishing, a full-length product captured by the magnetic beads is separated, the full-length product is subjected to first-round amplification and purification through Read1 and a universal joint primer, and the space-time transcriptome cDNA library products are obtained; and performing a second round of amplification and purification on the target sequence subjected to the first round of amplification to obtain a space-time transcriptome specific enriched product. When the method is applied to immune group analysis, the space gene information of the whole tissue slice is reserved, and meanwhile, the CDR3 regions of the BCR and TCR sequences of the immune group can be specifically enriched, so that the distribution of various immune cell types in the tissue and the gene expression mode can be better researched. The method provided by the invention fills the vacancy of a spatial immune group, and provides a reliable basis for the research of immunomics.

Description

Technical Field

[0001] The invention belongs to the technical field of transcriptomes, and in particular relates to a method for spatiotemporal transcriptome specific enrichment and an application thereof. Background Art

[0002] Spatial transcriptomics (ST) is a cutting-edge technology that combines gene expression and spatial organization information, aiming to analyze the spatial distribution of cells and genes in tissues and their interactions. It breaks the limitations of traditional transcriptomics and provides an important tool for studying cell function, tissue structure and disease mechanisms by measuring RNA expression at specific spatial locations. For example, studying tumor heterogeneity and microenvironment characteristics; constructing gene expression maps in brain tissues to explore the functions of neurons and glial cells; analyzing the dynamic changes of gene expression during embryonic development; studying the repair mechanism after tissue damage, etc. Single-cell sequencing is a revolutionary technology that can analyze genome, transcriptome, epigenome and other information in single cells. It overcomes the problem of signal averaging in traditional population-level sequencing and is suitable for studying the heterogeneity and specific functions of cells in complex biological systems.

[0003] Specific enrichment of spatiotemporal transcriptomes aims to reveal the association between the spatiotemporal distribution of cells in tissues and gene expression. It combines the technologies of spatiotemporal transcriptomics and gene expression enrichment to provide detailed spatiotemporal dimension information for target gene sequences of interest. Through these technologies, scientists can study the spatiotemporal dynamic changes of genes in tissues at the single cell or single molecule level, providing a deeper understanding of disease mechanisms, tissue development, and the design of treatment strategies.

[0004] The core role of immunogenomics in modern biomedicine is becoming increasingly prominent. Its ability to combine multi-omics and high-throughput technologies has promoted the development of precision medicine and personalized treatment, and provided solutions to global health problems. For example, precision medicine designs personalized treatment plans through immunogenomic data (such as genotype, antibody spectrum, and immune cell composition). In cancer, treatment strategies are adjusted according to the patient's immune microenvironment (such as tumor-infiltrating lymphocyte types) to improve efficacy. Immunomics research has also made many important advances, bringing new ideas to basic research and clinical treatment. For example, the discovery of new immunotherapy targets, a variety of studies are committed to discovering new therapeutic targets, among which the integration of data across cancer types has revealed some protein targets with broad applicability. These targets may play a role in multiple tumor types, providing the possibility of developing new drugs and treatment models.

[0005] The immune receptors BCR (B cell receptor) and TCR (T cell receptor) are antigen receptors on the surface of B cells and T cells, respectively. The CDR3 region in their sequences is the core part of their antigen recognition, which is crucial to the function of the immune system and is produced by V(D)J gene rearrangement. CDR3 is located at the 3' end of the V region and the J region of the variable region of antibodies and TCRs, and can be found in the heavy and light chains of BCRs and the α and β chains of TCRs. This region provides the greatest contribution to the diversity of BCRs and TCRs due to the imprecision of V(D)J recombination, the addition / deletion of nucleotides in the joining region, and the diverse combinations. The dynamic changes in the CDR3 sequence can be used as an important indicator to evaluate the efficacy of vaccines and guide vaccine design. It can also be used as a biomarker for early diagnosis of diseases and monitoring of treatment effects.

[0006] Commercial products and solutions in the field of single-cell immunogenomics cover all aspects from sample processing to data analysis, promoting the rapid development of basic research and clinical applications. Commercial platforms include 10x Genomics, Singleron GEXSCOPE®, and BD Rhapsody. However, the spatial immunogenomics is still a blank. The significance of developing spatial immunogenomics technology lies in combining spatial information with the single-cell resolution of immunogenomics, providing unprecedented possibilities for in-depth understanding of the complex relationship between the function of the immune system, tissue microenvironment and disease. At the spatial resolution level, traditional immunogenomics technology usually lacks spatial information, while spatial immunogenomics technology can retain the in situ spatial position of immune cells and help study the interaction between immune cells and their microenvironment.

[0007] Currently, the only commercial products are single-cell immunogenomics-related technologies, and spatial immunogenomics products are still vacant. The spatial immunogenomics can not only explore the unique functions of different immune cell subsets in health and disease, that is, immune heterogeneity. It can also combine spatial information to study the distribution of immune cells in tissues and their relationship with pathological states. It can be applied to immune cell tracking in tumor microenvironment (TME) and infectious diseases, which is a current research hotspot. In view of this, the development of a method based on spatiotemporal transcriptome-specific enrichment has important application value. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention aims to provide a method for spatiotemporal transcriptome specific enrichment and its application. The method for spatiotemporal transcriptome specific enrichment can help researchers to more comprehensively understand the distribution of immune cells in tissues and their relationship with pathological states, especially in tumor tissues.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for spatiotemporal transcriptome specific enrichment, the method comprising the following steps:

[0011] (S1) Preparation of cDNA library: enriching the spatiotemporal transcriptome cDNA library product to obtain a cDNA library; the library structure of the spatiotemporal transcriptome cDNA library product is Read1-NNNN...NN-UMI-TTTT...TTVNNNN...NNNN-strand displacement adapter;

[0012] (S2) Sequence blocking and primer hybridization: blocking with cDNA library as template and hybridization with biotin-modified capture primers;

[0013] (S3) Magnetic bead fishing and magnetic bead washing: streptavidin magnetic beads capture the full-length product of the target sequence; then wash the magnetic beads carrying the target product;

[0014] (S4) Full-length product amplification and purification: The full-length product captured by the magnetic beads was amplified and purified using Read1 and strand displacement adapter primers;

[0015] (S5) Second round of amplification and purification: The target sequence is subjected to a second round of amplification and purification to obtain spatiotemporal transcriptome-specific enrichment products.

[0016] The library structure of the spatiotemporal transcriptome cDNA library product in the present invention is Read1-NNNN...NN-UMI-TTTT...TTVNNNN...NNNN-strand displacement adapter; the spatiotemporal transcriptome cDNA library structure on the market is basically this structure. Among them, Read1: sequencing primer, NNN...NN-UMI: barcode information and molecular label, TTTT...TTVN: capture sequence, NNN...NNNN: gene sequence, strand displacement adapter: 5' universal adapter primer added during reverse transcription.

[0017] The method of the present invention is a specific enrichment scheme designed for spatiotemporal transcriptomes. Currently, the library structure of spatiotemporal transcriptomes on the market is "Read1-NNNN...NN-UMI-TTTT...TTVNNNN...NNNN-chain displacement adapter". The scheme of the present invention is particularly suitable for libraries of spatiotemporal transcriptome type.

[0018] In a specific embodiment of the present invention, Qingdao Baichuang S series spatial transcriptome products, DG1000 single-cell transcriptome products and 10x Genomics single-cell transcriptome products are used for specific enrichment.

[0019] In the present invention, the capture primers used are biotin-modified and designed in the conserved region of the desired specific enrichment sequence, and the hybridization temperature thereof needs to be determined according to the Tm value of the designed primers.

[0020] Preferably, in (S2), the hybridization temperature is 60-70°C, for example, 60°C, 62°C, 64°C, 65°C, 66°C, 68°C or 70°C, and the time is 1-14 h, for example, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h or 14 h.

[0021] In the present invention, the capture primer is designed based on the conserved region of the immune group BCR and TCR. In a specific implementation case of the present invention, the C region primer of the BCR and the C region primer of the TCR are used. The capture primer used in the present invention has the property of biotin modification to facilitate subsequent capture.

[0022] Preferably, in (S2), the biotin-modified capture primer is designed based on the conserved region of the target sequence.

[0023] Preferably, in (S2), the blocking comprises using the reverse complementary sequence of the fixed linker sequence in the library or the Cot1-DNA sequence to block the repetitive DNA sequence in the library, thereby achieving the purpose of reducing non-specific hybridization.

[0024] Preferably, in (S3), the streptavidin magnetic beads further include a step of washing with a washing buffer before use.

[0025] Preferably, in (S3), the capturing temperature is 60-70°C, for example, 60°C, 62°C, 64°C, 65°C, 66°C, 68°C or 70°C, and the capturing time is 0.5-1 h, for example, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h or 1 h.

[0026] In the present invention, the capture temperature is 60°C-70°C (the same as the hybridization temperature above), and the time is 30 minutes-1 hour, which can be adjusted according to the effect. The above temperature ensures that the probe and the target sequence can still be specifically bound during the magnetic bead capture, preventing the probe from separating or non-specifically hybridizing with other non-target sequences.

[0027] Preferably, in (S3), the washing includes hot washing and normal temperature washing, the temperature of the hot washing is 60-70°C, for example, it can be 60°C, 62°C, 64°C, 65°C, 66°C, 68°C or 70°C, etc., and the temperature of the normal temperature washing is 20-25°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, etc.

[0028] In the present invention, the hot wash is to initially wash away the non-specific target sequences at the hybridization temperature, and the normal temperature wash is to further wash away the non-specific and redundant capture probes.

[0029] Preferably, in (S5), a second round of amplification is performed using Read1 and an upstream primer encompassing the target sequence.

[0030] In the present invention, the upstream primers covering the target sequence refer to the enrichment primers artificially designed for the target sequence to be studied. For example, the case immune group of the present invention is a series of primers designed for the 5-terminal variable region (V region) of BCR and TCR.

[0031] Preferably, the second round of amplification includes any one of singleplex amplification, multiplex amplification or nested amplification.

[0032] In a second aspect, the present invention provides application of the method for spatiotemporal transcriptome specific enrichment described in the first aspect in immunomics.

[0033] It should be noted that the application of the present invention is not intended for the diagnosis or treatment of a disease. If the application of the present invention involves the diagnosis or treatment of a disease, the technical solution in this case is not within the scope of protection of the present invention.

[0034] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a spatiotemporal transcriptome-specific enrichment method and its application in the immune group. It combines the techniques of spatiotemporal transcriptomics and gene expression enrichment, and can provide detailed spatiotemporal dimension information for an in-depth understanding of the target gene sequence. In the process of spatiotemporal transcriptome analysis, the method provided by the present invention is used to deeply analyze the cellular heterogeneity, dynamic changes and functional characteristics of the immune system in the single-cell dimension, with a single cell as the resolution unit. In the spatial dimension, while retaining the spatial gene information of the entire tissue section, the CDR3 of the BCR and TCR sequences of immune cells can also be specifically enriched, thereby better studying the distribution of immune cells in tissues and their relationship with pathological states. The method provided by the present invention provides a reliable technical method for the scheme of spatiotemporal transcriptome-specific enrichment, and the application case in the present invention fills the gap in the spatial immune group and provides a reliable basis for the study of the immune group. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 15400 quality control of BCR heavy chain product fragments in spatial transcriptome human hepatocellular carcinoma tissue.

[0038] Figure 2 It is a 5400 quality inspection of BCR light chain product fragments in human liver cancer tissues of the Baichuang S series spatial transcriptome.

[0039] Figure 3 It is the 5400 quality inspection of TCRα chain product fragments in human liver cancer tissues of the Baichuang S series spatial transcriptome.

[0040] Figure 4 It is a 5400 quality inspection of BCRβ chain product fragments in human liver cancer tissues of the Baichuang S series spatial transcriptome.

[0041] Figure 5 5400 QC of BCR heavy chain product fragments in 10×Genomics single cell transcriptome PBMC samples.

[0042] Figure 6 5400 QC of BCR light chain product fragments in 10×Genomics single cell transcriptome PBMC samples.

[0043] Figure 7 5400 QC of TCRα chain product fragments in 10×Genomics single cell transcriptome PBMC samples.

[0044] Figure 8 5400 QC of TCRβ chain product fragments in 10×Genomics single cell transcriptome PBMC samples.

[0045] Fig. 9 This is a 5400 quality check of BCR heavy chain product fragments in the Biochuang DG1000 single cell transcriptome thyroid cancer sample.

[0046] Fig.10 This is a 5400 quality check of BCR light chain product fragments in the Baichuang DG1000 single cell transcriptome thyroid cancer sample.

[0047] Fig.11 This is a 5400 quality check of TCRα chain product fragments in the Baichuang DG1000 single cell transcriptome thyroid cancer sample.

[0048] Fig.12 This is a 5400 quality check of TCRβ chain product fragments in the Biochuang DG1000 single cell transcriptome thyroid cancer sample.

[0049] Fig.13 This is the clonal type distribution map of CDR3 of BCR in liver cancer tissue section of the example sample at the 100 μm spot level.

[0050] Fig.14 This is the clonal distribution map of CDR3 of TCR in liver cancer tissue section of the example sample at the 100 μm spot level.

[0051] Fig.15 It is a schematic diagram of the technical process of the present invention. DETAILED DESCRIPTION

[0052] The present invention provides a method based on spatiotemporal transcriptome specific enrichment. In order to verify this method, the present invention is based on the total cDNA product of the spatiotemporal transcriptome, combined with the immunogroup technology, to capture and enrich the immune group BCR and TCR products in the total product and enrich the CDR3 in the BCR and TCR sequences. The fragment distribution is detected by Agilent 5400, and the target sequence is compared by means of third-generation sequencing, and the results are stable and reliable.

[0053] The specific implementation plan is as follows:

[0054] (1) Primer design

[0055] Hybridization primers for human immune group were designed in the C region of immune group sequence, with about 200 BCRs and about 50 TCRs. About 80 BCR enrichment primers and about 100 TCR enrichment primers were designed in the V region.

[0056] (2) cDNA sample preparation

[0057] Prepare the total cDNA products of the spatiotemporal transcriptome of human samples, amplify and purify them by PCR to obtain a high-concentration cDNA library.

[0058] (3) Sequence blocking and primer hybridization

[0059] First, add the cDNA sample to the hybridization premix system, let it stand at room temperature for 5 minutes, and incubate it at 95°C for 10 minutes; then add the hybridization capture primers and incubate it at 68°C for 1 hour.

[0060] (4) Cleaning of streptavidin magnetic beads

[0061] The magnetic beads were washed with magnetic bead washing buffer, resuspended with hybridization premix, and stored at 68°C for later use.

[0062] (5) Fishing with streptavidin magnetic beads

[0063] Add all the hybridization system to the resuspended magnetic beads and incubate at 68°C for 1 h.

[0064] (6) Magnetic bead cleaning after fishing

[0065] After the incubation, the magnetic beads were washed with washing buffer at 68°C and room temperature. After the washing was completed, the residual washing buffer was discarded and the magnetic beads were resuspended with 20 μL of enzyme-free water.

[0066] (7) Full-length product amplification and purification

[0067] The product with magnetic beads was PCR amplified using Read1 and universal adapter primers, and the amplified product was purified using SPRI magnetic beads (0.6×).

[0068] (8) Multiple amplification, purification and quality inspection

[0069] 50 ng of BCR full-length product was taken to enrich BCR heavy chain and light chain, and 50 ng of TCR full-length product was taken to enrich TCR α chain and β chain. The amplified products were purified using SPRI magnetic beads (0.6×). The purified products were subjected to fragment distribution quality inspection using 5400. The results are shown in the table. Figure 1-Figure 12 .

[0070] (9) Third-generation library construction and sequencing: Construct the PacBio Hifi library and perform sequencing.

[0071] (10) Third-generation data analysis: PacBio's Sequel II platform was used to obtain third-generation full-length sequencing data. The BST Matrix tool independently developed by Biochuang was used to retain reads that identified barcodes (cell unique identifiers) and UMIs (molecular unique identifiers). IgBlast (version v1.22.0) was used to detect the light and heavy chain information of B cell receptors (BCR) and T cell receptors (TCR). At the same time, the barcodes (cell unique identifiers) corresponding to these reads were integrated at different resolutions to obtain immune group data at different resolutions. Spatial samples can combine data of different resolutions and cell segmentation levels, organize CDR3 data into expression matrix form, and use the Seurat package in the R environment for spatial display, thereby revealing the distribution characteristics of clonal types in different cell populations.

[0072] This approach not only allows us to accurately depict the distribution of immune cells in tissues, but also to gain a deep understanding of their potential roles in immune responses. This provides us with a comprehensive and detailed perspective to explore and interpret the complexity of immune cells.

[0073] The method for specific enrichment of spatiotemporal transcriptomes provided by the present invention has the advantages of novel results (there is no precedent for spatial immunogroups in the market) and stable results (reliable results can be output when testing a variety of cancer tissue samples).

[0074] In this invention, the study of immunogenomics is taken as an example. The core is to analyze the cellular heterogeneity, dynamic changes and functional characteristics of the immune system in a single cell unit through the spatiotemporal immunogenomic method. The spatial gene information of the entire tissue section is retained, and the BCR and TCR sequences of human immune cells are specifically enriched. This method is a general means, not limited to the cases listed.

[0075] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0076] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0077] Example 1

[0078] This embodiment provides a method for spatiotemporal immunity group, comprising the following steps:

[0079] 1. Primer design

[0080] The capture primers, multiple enrichment primers and other primer sequences involved in this example are shown in Table 1.

[0081] Table 1

[0082]

[0083] 2. cDNA Sample Preparation

[0084] 2.1 Take 50 ng of the cDNA product of the Biotron S series spatial transcriptome of human liver cancer tissue samples, 50 ng of the cDNA product of the 10xGenomics single cell transcriptome of human PBMC samples, and 50 ng of the cDNA product of the Biotron DG1000 single cell transcriptome of human thyroid cancer samples, prepare them according to the following amplification system, perform PCR amplification for 8 cycles, and measure the concentration. Set the temperature according to Table 2 below.

[0085] Table 2

[0086]

[0087] 2.3 Purify with SPRI magnetic beads (0.8×) and dissolve in 15 μL EB to obtain a cDNA library that meets the concentration requirements.

[0088] 3. Sequence blocking and primer hybridization

[0089] 3.1 Set the temperature according to Table 3 below.

[0090] Table 3

[0091]

[0092] 3.2 Prepare the premixed hybridization system according to Table 4 below (here, prepare two sets for each sample to capture BCR and TCR respectively).

[0093] Table 4

[0094]

[0095] 3.3 Add 3.5 μL cDNA to each of the two premixed hybridization systems, shake and mix, let stand at room temperature for 5 min, and then incubate at 95°C for 10 min.

[0096] 3.4 After incubation, take out the samples from the PCR instrument, add 1 μL BCR capture primer to one group of each sample and 1 μL TCR capture primer to the other group, and incubate at 68℃ for 1 h.

[0097] 4. Clean the streptavidin magnetic beads before use

[0098] 4.1 The streptavidin magnetic beads need to be equilibrated at room temperature for more than half an hour in advance.

[0099] 4.2 Dilute the magnetic bead washing buffer 1 to 1× working solution concentration and keep at room temperature for later use.

[0100] 4.3 Prepare magnetic bead resuspension solution for each reaction according to Table 5 below and keep at room temperature for later use.

[0101] Table 5

[0102]

[0103] 4.4 For each reaction, take 50 μL of streptavidin magnetic beads into a PCR tube, adsorb on a magnetic stand, and discard the supernatant after clarification.

[0104] 4.5 Add 100 μL of streptavidin magnetic bead washing buffer 1 respectively, pipette to mix, adsorb on a magnetic stand, and discard the supernatant after clarification.

[0105] 4.6 Repeat step 4.5 and wash three times.

[0106] 4.7 Use the magnetic bead resuspension solution prepared in 4.3 to resuspend the streptavidin magnetic beads, being careful not to create bubbles.

[0107] 5. Fishing with streptavidin magnetic beads

[0108] 5.1 Preheat the resuspended streptavidin beads at 68°C in a shaking incubator.

[0109] 5.2 After the incubation, take out the hybridization products from the PCR instrument and place them in a 68℃ shaking incubator. Transfer all the hybridization products to the resuspended streptavidin magnetic beads, pipette and mix well, and incubate at 68℃ for 1 h at 1500 rpm.

[0110] 6. Cleaning of Streptavidin Magnetic Beads after Fishing

[0111] 6.1 Reagent preparation: Prepare washing buffer 2, buffer 3, buffer 4, and buffer 5 to a concentration of 1× for standby use. Buffer 2 and buffer 3 required for hot washing should be preheated to 68°C in advance for at least 15 minutes. (The above buffers were purchased from Qingdao Baichuang Intelligent Manufacturing Biotechnology Co., Ltd.)

[0112] 6.2 Hot washing of magnetic beads

[0113] 6.2.1 After capture, add 100 μL of preheated buffer 3 to each PCR tube, mix gently by pipetting, adsorb on a magnetic rack, clarify, and discard the supernatant.

[0114] 6.2.2 Add 150 μL of preheated buffer 2, mix by gentle pipetting, incubate at 68°C for 5 min, adsorb on a magnetic rack, and discard the supernatant after clarification.

[0115] 6.2.3 Repeat 6.2.2.

[0116] 6.3 Washing magnetic beads at room temperature

[0117] 6.3.1 Add 150 μL of room temperature buffer 3 to each PCR tube, shake and mix for 2 min, adsorb on a magnetic rack, clarify, and discard the supernatant.

[0118] 6.3.2 Add 150 μL of room temperature buffer 4 to each PCR tube, shake and mix for 2 min, adsorb on a magnetic rack, clarify, and discard the supernatant.

[0119] 6.3.3 Add 150 μL of room temperature buffer 5 to each PCR tube, shake and mix for 2 min, adsorb on a magnetic rack, clarify, and discard the supernatant.

[0120] 6.3.4 Discard the remaining buffer, add 20 μL of enzyme-free water, and vortex to mix.

[0121] 7. Full-length product amplification and purification

[0122] 7.1 Prepare the amplification system on ice for each reaction according to Table 6 below.

[0123] Table 6

[0124]

[0125] 7.2 Set up the amplification program for each reaction according to Table 7 below

[0126] Table 7

[0127]

[0128] 7.3 Take 2 μL of streptavidin magnetic beads with product for PCR amplification, amplify for 15-20 cycles, and measure the concentration.

[0129] 7.4 The amplified products were purified using SPRI magnetic beads (0.6×), and the concentration was measured to obtain the pre-amplified products of BCR and TCR.

[0130] 8. Multiplex amplification, purification and quality inspection

[0131] 8.1 Prepare the amplification system on ice for each reaction according to Table 8-11 below.

[0132] Table 8

[0133]

[0134] Table 9

[0135]

[0136] Table 10

[0137]

[0138] Table 11

[0139]

[0140] 8.2 Set up the amplification program according to Table 12 below

[0141] Table 12

[0142]

[0143] 8.3 Amplify the BCR heavy chain and light chain separately as described above, measure the concentration, purify the amplified products using SPRI magnetic beads (0.6×), and measure the concentration to obtain BCR light and heavy chain enriched products.

[0144] 8.4 Amplify TCR α and β chains separately as described above, measure concentration, purify the amplified products using SPRI magnetic beads (0.6×), and measure concentration to obtain TCR α and β chain enriched products.

[0145] 8.5 Use Agilent 5400 to perform fragment distribution quality check. The results are shown in Figure 1-Figure 12 .

[0146] Figure 1-Figure 4The heavy and light chains of BCR, and the α and β chains of TCR enriched in the cDNA products of the Biotron S series spatial transcriptome of human liver cancer tissue samples; Figure 5-Figure 8 The heavy and light chains of BCR and the α and β chains of TCR enriched from the cDNA products of 10×Genomics single-cell transcriptome of human PBMC samples; Figure 9-12 These are the heavy and light chains of BCR, and the α and β chains of TCR enriched in the cDNA products of the single-cell transcriptome of the human thyroid cancer sample BioChuang DG1000.

[0147] From the results of quality inspection Figure 1-Figure 12 It can be seen that the length of the immune group products enriched by this method is stable and reproducible.

[0148] 9. Third-generation library construction and sequencing

[0149] The Hifi library was constructed using the SMRTbell Prep Kit 3.0 and the SMRT Cell 8M was used.

[0150] 10. Three-generation data analysis

[0151] PacBio's Sequel II platform is used to obtain third-generation full-length sequencing data. The BST Matrix tool independently developed by Biochuang can effectively extract the barcode (as the unique identifier of the cell) and UMI (as the unique molecular label) information in the full-length sequencing reads (read segments). In this process, only those reads that can identify both the barcode and UMI are retained, and those that cannot be identified are discarded.

[0152] Next, IgBlast (version v1.22.0) was used to detect the light and heavy chain information of B cell receptor (BCR) and T cell receptor (TCR). At the same time, the barcodes corresponding to these reads were integrated at different resolutions to obtain immune group data at different resolutions.

[0153] The core goal of spatiotemporal immunogenomics analysis is to explore the distribution changes of clonotypes (CDR3) in the spatial location of tissues. Combining data of different resolutions and cell segmentation levels, CDR3 data is organized into an expression matrix for more in-depth downstream analysis. For example, the Seurat package can be used in the R environment for spatial display to reveal the distribution characteristics of clonotypes in different cell populations.

[0154] This approach not only allows for a precise depiction of the distribution of immune cells in tissues, but also a deep understanding of their potential roles in immune responses. This provides a comprehensive and detailed perspective to explore and interpret the complexity of immune cells.

[0155] The data of the space immunity group is shown in Table 13 as an example.

[0156] Table 13

[0157]

[0158] In Table 13, the type description is as follows:

[0159] Reads Mapped to Any V(D)J Gene: The proportion of sequencing fragments that are partially or fully mapped to any V(D)J gene segment.

[0160] Reads Mapped to IGH: The proportion of sequencing fragments that are partially or fully mapped to the IGH gene segment.

[0161] Reads Mapped to IGK: The proportion of sequencing fragments that are partially or fully mapped to the IGK gene segment.

[0162] Reads Mapped to IGL: The proportion of sequencing fragments that are partially or fully mapped to the IGL gene segment.

[0163] Reads With CDR3-annotated IGH: The proportion of sequencing fragments with CDR3 detected on IGH.

[0164] Reads With CDR3-annotated IGK: The proportion of sequencing fragments with CDR3 detected on IGK.

[0165] Reads With CDR3-annotated IGL: The proportion of sequencing fragments with CDR3 detected on IGL.

[0166] Reads With Productive IGH: The proportion of sequenced fragments with biological functions on IGH.

[0167] Reads With Productive IGK: The proportion of sequencing fragments with biological functions on IGK.

[0168] Reads With Productive IGL: The proportion of sequenced fragments with biological functions on IGL.

[0169] Productive: There is a start codon in the expected part of the V sequence, an in-frame CDR3, and no stop codon in the aligned VJ region.

[0170] As can be seen from Table 13, the BCR gene comparison rate results are ideal.

[0171] The TCR enrichment is shown in Table 14:

[0172] Table 14

[0173]

[0174] In Table 14, the type description is as follows:

[0175] Reads Mapped to Any V(D)J Gene: The proportion of sequencing fragments that are partially or fully mapped to any V(D)J gene segment.

[0176] Reads Mapped to TRA: The proportion of sequencing fragments that are partially or fully mapped to the TRA gene segment.

[0177] Reads Mapped to TRB: The proportion of sequencing fragments that are partially or fully mapped to the TRB gene segment.

[0178] Reads With CDR3-annotated TRA: The proportion of sequencing fragments with CDR3 detected on TRA.

[0179] Reads With CDR3-annotated TRB: The proportion of sequencing fragments with CDR3 detected on TRB.

[0180] Reads With Productive TRA: The proportion of sequenced fragments with biological functions on TRA.

[0181] Reads With Productive TRB: The proportion of sequenced fragments with biological functions on TRB.

[0182] Productive: There is a start codon in the expected part of the V sequence, an in-frame CDR3, and no stop codon in the aligned VJ region.

[0183] As can be seen from Table 14, the TCR gene matching rate results are ideal.

[0184] Analysis at different resolution dimensions, including: L2, L7 and L13.

[0185] The BCR enrichment is shown in Table 15:

[0186] Table 15

[0187]

[0188] Description of the types in Table 15:

[0189] IGH Contig: The proportion of at least one IGH annotated as a complete or partial V(D)J gene.

[0190] IGK Contig: The proportion of at least one IGK annotated as a complete or partial V(D)J gene.

[0191] IGL Contig: The proportion of at least one IGL annotated as a complete or partial V(D)J gene.

[0192] CDR3-annotated IGH Contig: The proportion of IGH with at least one detectable CDR3.

[0193] CDR3-annotated IGK Contig: The proportion of IGKs with at least one detectable CDR3.

[0194] CDR3-annotated IGL Contig: The proportion of IGLs with at least one detectable CDR3.

[0195] Productive IGH Contig: At least one contig spans from the 5' end of the V region to the 3' end of the J region of IGH, has a start codon in the expected part of the V sequence, has an in-frame CDR3, and has no stop codon in the aligned VJ regions.

[0196] Productive IGK Contig: At least one spanning the 5' end of the V region to the 3' end of the J region of IGK, with a start codon in the expected part of the V sequence, an in-frame CDR3, and no stop codon in the aligned VJ regions.

[0197] Productive IGL Contig: at least one IGL spanning from the 5' end of the V region to the 3' end of the J region, with a start codon in the expected part of the V sequence, an in-frame CDR3, and no stop codon in the aligned VJ regions.

[0198] Productive VJ Spanning Pair: The proportion of receptor pairs in which at least one chain of each chain is functional.

[0199] Productive VJ Spanning (IGK, IGH) Pair: The proportion of (IGK, IGH) receptor pairs in which at least one of each chain is functional.

[0200] Productive VJ Spanning (IGL, IGH) Pair: The proportion of (IGL, IGH) receptor pairs in which at least one of each chain is functional.

[0201] Productive: The annotation spans the 5' end of the V region to the 3' end of the J region of the chain, a start codon is present in the expected part of the V sequence, an in-frame CDR3 amino acid domain is present, and a stop codon is absent in the aligned VJ regions.

[0202] As can be seen from Table 15, the detection ratio of CDR3 of BCR is ideal.

[0203] The TCR enrichment is shown in Table 16:

[0204] Table 16

[0205]

[0206] Description of the types in Table 16:

[0207] TRA Contig: The proportion of at least one TRA annotated as a complete or partial V(D)J gene.

[0208] TRB Contig: The proportion of at least one TRB annotated as a complete or partial V(D)J gene.

[0209] CDR3-annotated TRA Contig: The proportion of TRA with at least one detectable CDR3.

[0210] CDR3-annotated TRB Contig: The proportion of TRBs with at least one detectable CDR3.

[0211] Productive IGH contigs: at least one spans the 5' end of the V region to the 3' end of the J region of the TRA, has a start codon in the expected part of the V sequence, has an in-frame CDR3, and has no stop codon in the aligned VJ regions.

[0212] Productive IGK contigs: at least one V region spanning the 5' end of the TRB to the 3' end of the J region, with a start codon in the expected part of the V sequence, an in-frame CDR3, and no stop codon in the aligned VJ regions.

[0213] Productive VJ Spanning Pair: The proportion of receptor pairs in which at least one chain of each chain is functional.

[0214] Productive VJ Spanning (TRA, TRB) Pair: The proportion of (TRA, TRB) receptor pairs in which at least one of each chain is functional.

[0215] Productive: The annotation spans the 5' end of the V region to the 3' end of the J region of the chain, a start codon is present in the expected part of the V sequence, an in-frame CDR3 amino acid domain is present, and a stop codon is absent in the aligned VJ regions.

[0216] As can be seen from Table 16, the CDR3 detection ratio of TCR is ideal.

[0217] In summary, the present invention provides a method for specific enrichment of spatiotemporal transcriptomes, and applies the method to specifically enrich immune group sequences. The method of the present invention fills the gap in spatial immune groups and provides a reliable basis for the study of immunogenomics.

[0218] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for spatiotemporal transcriptome specific enrichment, characterized in that: The method comprises the following steps: (S1) Preparation of cDNA library: enriching the spatiotemporal transcriptome cDNA library product to obtain a cDNA library; the library structure of the spatiotemporal transcriptome cDNA library product is Read1-NNNN...NN-UMI-TTTT...TTVNNNN...NNNN-strand displacement adapter; (S2) Sequence blocking and primer hybridization: blocking with cDNA library as template and hybridization with biotin-modified capture primers; (S3) Magnetic bead fishing and magnetic bead washing: Streptavidin magnetic beads capture the full-length product of the target sequence; Then wash the magnetic beads carrying the target product; (S4) Full-length product amplification and purification: The full-length product captured by the magnetic beads was amplified and purified using Read1 and universal adapter primers; (S5) Second round of amplification and purification: The target sequence is subjected to a second round of amplification and purification to obtain spatiotemporal transcriptome-specific enrichment products.

2. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: (S2), the hybridization temperature is 60-70°C and the time is 1-14 h.

3. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: In (S2), the biotin-modified capture primer is designed based on the conserved region of the target sequence.

4. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: In (S2), the blocking includes using the reverse complementary sequence of the fixed linker sequence in the library or the Cot1-DNA sequence to block the repetitive DNA sequence in the library, thereby achieving the purpose of reducing non-specific hybridization.

5. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: In (S3), the streptavidin magnetic beads also include a step of washing with a washing buffer before use.

6. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: In (S3), the capture temperature is 60-70°C and the capture time is 0.5-1 h.

7. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: In (S3), the washing includes hot washing and normal temperature washing, the temperature of the hot washing is 60-70°C, and the temperature of the normal temperature washing is 20-25°C.

8. The method for spatiotemporal transcriptome specific enrichment according to claim 1, characterized in that: In (S5), a second round of amplification is performed using Read1 and an upstream primer encompassing the target sequence.

9. The method for spatiotemporal transcriptome specific enrichment according to claim 8, characterized in that: The second round of amplification includes any one of single-plex amplification, multiplex amplification or nested amplification.

10. Use of the method for spatiotemporal transcriptome specific enrichment according to any one of claims 1 to 9 in immunomics.

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