TCR beta chain enrichment method

Abstract

The invention discloses a method for enriching a TCR (T cell receptor) beta chain. The method comprises the following steps: hybridizing a probe group and a to-be-detected sample, wherein the to-be-detected sample comprises a nucleotide sequence of a TCR beta chain; and collecting a hybridization treatment product to obtain the TCR beta chain, the probe group comprises one or more probes, and the probes are combined with a constant region of a TCR beta chain. According to the method, the TCR beta chains in the sample to be detected can be effectively enriched, the enriched TCR beta chains are used for sequencing, and high-abundance TCR full-length sequence information can be obtained; moreover, space-time transcriptome sequencing is carried out on the enriched TCR beta chain, so that the UMI capture number of the TCR beta chain can be increased, and the abundance of the TCR beta chain can also be increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene sequencing. Specifically, the present invention relates to a method for enriching TCR beta chains, and more specifically, the present invention relates to a spatial and temporal transcriptome sequencing method based on TCR beta chain enrichment. Background Art

[0002] The T cell receptor (TCR) is a protein on the surface of T cells responsible for specifically recognizing antigen peptides bound to the major histocompatibility complex (MHC). When TCR binds to antigen peptides and MHC, T lymphocytes are activated through signal transduction and participate in the body's immune response process. TCR can be divided into a constant region (C region) and a variable region (V region) according to its gene and transcript sequence. Among them, the constant region is close to the 3' end with a length of about 400 bp; while the variable region is located at the 5' end and contains three framework regions (FR1 / 2 / 3) and two complementary determining regions (CDR1 / 2). The 3' end of the variable region and the subsequent closely connected (D)J region together form the complementary determining region 3 (CDR3), and the sequence of CDR3 directly determines the affinity of TCR for antigen polypeptides. TCR consists of two chains, an alpha chain and a beta chain (heterodimer), and undergoes V(D)J gene rearrangement in the thymus to form diverse T cell clones. The diversity of TCR is the basis for the body to recognize various antigens.

[0003] After T cells undergo TCR recombination and antigen selection processes, they play an immune surveillance function in various organs through the lymphatic and blood circulatory systems. The TCR sequences and clone abundances in a specific region are called the immune repertoire. Currently, immune repertoire sequencing technology is often used to identify T cells and their homologous TCR. This method usually relies on in vitro T cell function assays to obtain DNA or RNA fragments from body T cells, and based on multiplex PCR or 5'RACE technology (see Figure 1 ), target amplify the CDR3 region of the beta chain or the full-length V(D)J sequence, and then construct a library for sequencing. However, whether it is multiplex PCR or 5'RACE technology, the obtained TCR beta chain abundance is low, and it is impossible to more conveniently obtain higher abundance TCR full-length sequences and unknown sequences from single-cell transcriptome samples, nor can it be combined with spatial and temporal transcriptome technology, which has great limitations in solving the research on the spatial distribution of TCR-T. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this reason, the present invention provides a method for enriching TCR beta chains.

[0005] The present invention is completed based on the following discoveries of the inventors:

[0006] At present, although two mainstream TCR-seq technologies (multiplex PCR or 5' RACE technology) have corresponding applications in the field of single-cell transcriptomics, both still have corresponding disadvantages and cannot complement each other.

[0007] The TCR-seq technology based on the multiplex PCR strategy mainly designs primers based on known V-region allele sequences and cannot detect unknown new V-allele mutations; secondly, PCR amplification based on primers cannot avoid PCR amplification bias, and the primers compete, affecting the relative abundance of products and unable to distinguish PCR duplicates from molecular duplicates; thirdly, multiplex PCR can only amplify the CDR3 region and cannot obtain the full-length TCR information; fourthly, the sequencing of the products of the multiplex PCR strategy is population sequencing, and the diversity of TCR cannot be analyzed at the single-cell level and spatial dimension.

[0008] The TCR-seq technology based on the 5' RACE strategy is only applicable to RNA samples for library construction and sequencing, and has high requirements for sample quality. This technology uses RNA as the starting material, and the enrichment of mRNA through nested PCR / circular PCR requires at least two rounds of PCR, and the process is cumbersome, which easily affects the relative abundance of products and experimental repeatability. Although the 5' RACE strategy can be applied to the single-cell transcriptome level for library construction and sequencing to obtain the full-length TCR sequence, at the spatial and temporal transcriptome level, it is impossible to use the cDNA products that have completed reverse transcription on the chip as the starting point for TCR enrichment and library construction and sequencing.

[0009] In addition, since most current spatial and temporal transcriptome technologies rely on poly T to capture the 3' poly A tail, limited by the read length of next-generation sequencing (≤300 bp), the spatial and temporal transcriptome often cannot directly obtain TCR sequences and the localization information of antigen-specific T cells, and the TCR-seq technologies based on multiplex PCR or 5' RACE technology cannot perform secondary library construction from the cDNA products captured by the spatial and temporal transcriptome and obtain the full-length TCR sequence containing spatial position information.

[0010] Based on this, the present invention designs a probe group for the constant region of the TCR beta chain, and the probe group can bind to the constant region of the TCR beta chain. The inventors found that the DNA probe designed for the TRBC gene sequence, DNA-DNA hybridization cannot effectively release the captured TCR beta chain, and the obtained product does not meet the requirements of sequencing library construction. Therefore, the inventors replaced the DNA probe designed for the TRBC gene sequence with an RNA probe, and modified the 5' end of the RNA probe to enrich the TCR beta chain, which has a relatively low proportion, thereby increasing the abundance of the acquired TCR beta chain. Furthermore, the above-mentioned probe group and cDNA samples are hybridized, amplified and sequenced to obtain a complete full-length sequence of the TCR beta chain, which is helpful to identify new V region allele mutations, especially the cDNA products captured from the spatiotemporal transcriptome can be used for secondary library construction and obtain the full-length sequence of the TCR beta chain containing spatial position information.

[0011] Therefore, in the first aspect of the present invention, the present invention proposes a probe group. According to an embodiment of the present invention, it includes one or more probes; wherein, the probe is RNA, and the probe can be complementary to at least part of the sequence of the constant region of the TCR beta chain. The probe group according to the embodiment of the present invention is combined with the constant region of the TCR beta chain, and can capture a large number of TCR beta chains that have a low proportion in the sample to be tested, thereby increasing the abundance of the TCR beta chain. The above-mentioned probe group is used to hybridize and capture cDNA samples, especially hybridize and capture cDNA samples obtained by stereo-seq, and the captured TCR beta chain is spatially transcripted and library-built and sequenced, which can not only increase the number of UMI captures of the TCR beta chain, but also increase the abundance of the TCR beta chain; in addition, the TCR beta chain library captured by the probe group is used for single-molecule sequencing, and the full-length sequence information of the high-abundance TCR beta chain can be obtained.

[0012] In a second aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit comprises: the probe group described in the first aspect of the present invention. The kit according to an embodiment of the present invention can capture a large number of TCR beta chains that have a relatively low proportion in the sample to be tested, thereby increasing the abundance of the TCR beta chain.

[0013] In the third aspect of the present invention, the present invention proposes a method for enriching TCR beta chain. According to an embodiment of the present invention, the method comprises: performing hybridization treatment on the probe group described in the first aspect of the present invention or the kit described in the second aspect of the present invention and a sample to be tested, wherein the sample to be tested contains a nucleotide sequence of the TCR beta chain; collecting the hybridization treatment product to obtain the TCR beta chain.

[0014] As described above, the probe set according to the first aspect of the present invention binds to the constant region of the TCR beta chain. By hybridizing the above probe set with a sample to be tested, the TCR beta chain in the sample to be tested can be captured. Thus, the method of the present invention can enrich a large number of TCR beta chains, especially capture the TCR beta chains with a relatively low proportion in the sample to be tested, thereby increasing the abundance of the obtained TCR beta chains. Further, performing spatial transcriptome sequencing on the enriched TCR beta chains can not only increase the UMI capture number of the TCR beta chains, but also increase the abundance of the TCR beta chains; in addition, using the TCR beta chain library obtained by enriching this method for single-molecule sequencing can obtain the full-length sequence information of the TCR beta chains with high abundance.

[0015] In the third aspect of the present invention, the present invention provides a method for sequencing TCR beta chains. According to an embodiment of the present invention, the method includes: hybridizing a sample to be tested with the probe set according to the first aspect of the present invention, wherein the sample to be tested contains the nucleotide sequence of the TCR beta chain; collecting the hybridization product to obtain the nucleotide sequence of the TCR beta chain; and constructing a library and sequencing the nucleotide sequence of the TCR beta chain. The sequencing method of the present invention can obtain the full-length sequence information of the TCR beta chains with high abundance.

[0016] In the fourth aspect of the present invention, the present invention provides a spatial transcriptome sequencing method. According to an embodiment of the present invention, the method includes: sequencing the TCR in a tissue sample to be tested using the aforementioned method; and obtaining the spatial transcriptome information of the tissue sample to be tested based on the sequencing results. The method of the present invention can increase the UMI capture number of the TCR beta chains, help to locate more TCR-Ts that could not be detected before, and obtain the full-length sequence information of the TCRs with high abundance.

[0017] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0019] Figure 1 It is the experimental principle diagram of multiplex PCR and 5'RACE technologies, as well as the schematic diagram of the existing TCR sequencing service (BGI Immunome Sequencing Service).

[0020] Figure 2Schematic diagram of the CHromium single-cell V(D)J kit developed for 10x Genomics, and schematic diagram of the single-cell full-length immune receptor kit of sCircleR.

[0021] Figure 3 Enrichment strategy of multiple probes in the embodiment solution of the present invention in Example 2.

[0022] Figure 4 Statistical chart of the capture rate ratio after enrichment using the solution of the present invention (Experimental groups 1-6 in Example 2) and that without using the solution of the present invention.

[0023] Figure 5 In Example 2, comparison chart of the capture rate ratio before and after enrichment of the TRBC gene in mouse lymph node samples, comparison chart of the consistency of the TCR copy spatial position in the follicular type (Tfh) region before and after enrichment; comparison chart of the capture rate ratio before and after enrichment of the TRBC gene in mouse colorectal cancer tumors.

[0024] Figure 6 In Example 2, comparison chart of the experimental results after enrichment treatment of the TRBC gene in human colorectal cancer tumor samples for Experimental group 6 and Experimental group 7. Detailed implementation manners

[0025] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0026] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0027] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0028] In this text, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0029] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may but do not necessarily occur, and this description includes the cases where such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0030] In this text, the term "spatial-temporal transcriptome" refers to an emerging transcriptomics technology aimed at revealing the spatial distribution of gene expression in tissues or cells. Traditional transcriptomics technologies usually provide the overall gene expression information of tissues or cells, but cannot provide the spatial information between cells. The spatial-temporal transcriptome technology fixes tissues or cells on tissue sections and performs high-throughput single-cell RNA sequencing on the sections, enabling the simultaneous acquisition of gene expression and spatial location information. The basic principle of the spatial-temporal transcriptome technology is to introduce spatial barcodes (Spatialbarcodes) on tissue sections, and these barcodes have specific spatial coordinates at different positions on the sections. Then, single-cell RNA sequencing technology is used to sequence the tissue sections, associating the gene expression information of cells with their spatial locations. In this way, the gene expression profile of each cell can be obtained, and the spatial distribution of genes between tissues or cells can be understood.

[0031] The present invention proposes a probe set, a method for enriching TCR beta chain, a method for TCR sequencing, and a spatial-temporal transcriptome sequencing method, which will be described in detail below respectively.

[0032] Probe set

[0033] In the first aspect of the present invention, the present invention proposes a probe set. According to an embodiment of the present invention, it includes one or more probes; wherein, the probe is RNA, and the probe can be at least partially complementary to the constant region sequence of the TCR beta chain. The probe set of the present invention can bind to the constant region of the TCR beta chain, thereby capturing the TCR beta chain, especially the TCR beta chain with a relatively low proportion in the sample to be tested, and can improve the abundance of the TCR beta chain. Further, the above probe set is used for hybridization capture of cDNA samples, especially for cDNA samples obtained by stereo-seq, and the captured TCR beta chain is subjected to spatial-temporal transcriptome library construction and sequencing, which can not only improve the UMI capture number of the TCR beta chain, but also improve the abundance of the TCR beta chain; in addition, the TCR beta chain library captured by the probe set is used for single-molecule sequencing, and high-abundance TCR full-length sequence information can be obtained.

[0034] According to an embodiment of the present invention, the probe binds to the TRBC gene in the constant region of the TCR beta chain.

[0035] According to an embodiment of the present invention, the TRBC gene in the constant region of the TCR beta chain is derived from a human or murine source.

[0036] According to an embodiment of the present invention, the probe has a nucleotide sequence as shown in any one of SEQ ID NO: 1 to 11.

[0037] According to an embodiment of the present invention, the probe set includes at least two probes.

[0038] According to an embodiment of the present invention, the probe set includes the nucleotide sequences as shown in SEQ ID NO: 1 to 5.

[0039] According to an embodiment of the present invention, the probe set includes the nucleotide sequences as shown in SEQ ID NO: 6 to 9, and the nucleotide sequences as shown in SEQ ID NO: 10 and / or SEQ ID NO: 11.

[0040] In one embodiment of the present invention, the probe set includes the nucleotide sequences as shown in SEQ ID NO: 6 to 10.

[0041] In one embodiment of the present invention, the probe set includes the nucleotide sequences as shown in SEQ ID NO: 6 to 9, 11.

[0042] According to an embodiment of the present invention, the 5' end of the probe has a first molecular modification.

[0043] According to an embodiment of the present invention, the first molecule includes at least one of biotin, globin, cyclic dimer, SNAP-tag, CLIP-tag or transcription factor tag.

[0044] In one embodiment of the present invention, the first molecule is biotin.

[0045] Method for enriching TCR beta chain

[0046] In a second aspect of the present invention, the present invention provides a method for enriching TCR beta chain. According to an embodiment of the present invention, the method includes: hybridizing the probe set described in the first aspect of the present invention with a sample to be tested, wherein the sample to be tested contains the nucleotide sequence of the TCR beta chain; collecting the hybridization product to obtain the TCR beta chain.

[0047] As can be seen from the above, the probe set according to the first aspect of the present invention binds to the constant region of the TCR beta chain. By hybridizing the above probe set with a sample to be tested, the TCR beta chain in the sample to be tested can be captured. Thus, the method of the present invention can enrich a large amount of TCR beta chains, especially TCR beta chains with a relatively low proportion in the sample to be tested, and can improve the abundance of the obtained TCR beta chains. Further, performing spatial transcriptome sequencing on the enriched TCR beta chains can not only increase the UMI capture number of the TCR beta chains, but also increase the abundance of the TCR beta chains; in addition, using the TCR beta chain library obtained by enriching this method for single-molecule sequencing can obtain high-abundance full-length TCR sequence information.

[0048] According to an embodiment of the present invention, the collection of the hybridization treatment product is carried out through the following steps: contacting the solid-phase carrier with the hybridization treatment product to obtain a solid-phase carrier connected with the hybridization treatment product; wherein, the solid-phase carrier is connected with a second molecule, and the second molecule binds to the first molecule of the probe.

[0049] According to an embodiment of the present invention, the first molecule includes at least one of biotin, globin, cyclic dimer, SNAP-tag, CLIP-tag, or transcription factor tag, and the second molecule includes at least one of streptavidin, Halo Tag conjugate, FIAsH / ReAsH dye, dye corresponding to SNAP-tag, dye corresponding to CLIP-tag, or recognition sequence corresponding to the transcription factor tag.

[0050] In the present invention, "globin" refers to an affinity tag obtained by protein engineering of Escherichia coli, which can stably bind to a Halo Tag conjugate (such as HaloLink Resin); "cyclic dimer" refers to a small tag composed of 4 cysteine residues, which can bind to fluorescent dyes such as FlAsH or ReAsH; "SNAP-tag" is an affinity tag modified from O6-demethylase (O6-alkylguanine-DNA alkyltransferase), which can bind to TMR-Star, 505-Star or 549; "CLIP-tag" is an affinity tag modified from O6-demethylase (O6-alkylguanine-DNA alkyltransferase), which can bind to TMR-Star, 505-Star or 549.

[0051] According to an embodiment of the present invention, the first molecule is biotin and the second molecule is streptavidin. The first molecule is globin and the second molecule is a Halo Tag conjugate. The first molecule is a cyclic dimer and the second molecule is a FIAsH / ReAsH dye. The first molecule is a SNAP-tag and the second molecule is a dye corresponding to the SNAP-tag. The first molecule is a CLIP-tag and the second molecule is a dye corresponding to the CLIP-tag. The first molecule is a transcription factor tag and the second molecule is an identification sequence corresponding to the transcription factor tag.

[0052] According to an embodiment of the present invention, the transcription factor tag includes, but is not limited to, at least one of LexA, Gal4, or VP16. Exemplarily, the identification sequence corresponding to Gal4 is 5'-CGG(N)_11-CCG-3', where N represents any nucleotide.

[0053] In one embodiment of the present invention, the first molecule is biotin and the second molecule is streptavidin.

[0054] According to an embodiment of the present invention, the solid-phase carrier includes at least one of magnetic beads, silica gel, affinity resin, ion exchange resin, and hydrophilic gel.

[0055] According to an embodiment of the present invention, it further includes: subjecting the solid-phase carrier connected with the hybridization treatment product to enzymatic digestion treatment to obtain the TCR beta chain.

[0056] According to an embodiment of the present invention, at least a partial sequence of the nucleotide sequence of the TCR beta chain is complementary and paired with at least a partial sequence of the probe in the probe set.

[0057] According to an embodiment of the present invention, the sample to be tested is a cDNA sample.

[0058] Method for TCR sequencing

[0059] In a third aspect of the present invention, the present invention provides a method for TCR sequencing. According to an embodiment of the present invention, the method includes: using the probe set described in the first aspect of the present invention to perform hybridization treatment on a sample to be tested, wherein the sample to be tested contains the nucleotide sequence of the TCR beta chain; collecting the hybridization treatment product to obtain the nucleotide sequence of the TCR beta chain; constructing a library and performing sequencing on the nucleotide sequence of the TCR beta chain. The sequencing method of the present invention can be applied to cDNA samples of single-cell transcriptome libraries to obtain high-abundance single-cell TCR beta chain sequence information without PCR bias, and the product can be directly used for second-generation / third-generation library construction and sequencing.

[0060] According to an embodiment of the present invention, the nucleotide sequence of the TCR beta chain is complementary paired with at least a partial sequence of the probe in the probe set.

[0061] As used herein, "at least a partial sequence complementary pairing" means that a part of the sequence in the probe (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) is complementary paired with the nucleotide sequence of the TCR beta chain. Among them, the sequences of the probe and the nucleotide sequence of the TCR beta chain that are complementary paired can be continuous or discontinuous.

[0062] According to an embodiment of the present invention, the nucleotide sequence is the full-length nucleotide sequence of the TCR beta chain.

[0063] According to an embodiment of the present invention, the nucleotide sequence is a cDNA sequence.

[0064] According to an embodiment of the present invention, the sample to be tested is a cDNA sample.

[0065] According to an embodiment of the present invention, the cDNA sample is obtained by successively performing a patching process, a tissue fixation process, a tissue permeabilization process, a reverse transcription process, a tissue removal process, and a cDNA release and recovery process on a section of the tissue sample to be tested.

[0066] It should be noted that the method for obtaining the cDNA sample is not specifically limited, as long as the cDNA sample can be obtained. In addition to the methods defined above, other acquisition methods are also within the protection scope of the present invention.

[0067] Exemplarily, the cDNA sample of the present invention can be obtained by the stereo-seq method.

[0068] According to an embodiment of the present invention, it further includes: after the cDNA release and recovery product, performing cDNA purification and amplification processing.

[0069] According to an embodiment of the present invention, the collection of the hybridization processing product is carried out through the following steps: contacting the solid phase carrier with the hybridization processing product to obtain a solid phase carrier connected with the hybridization processing product;

[0070] Among them, the solid carrier is connected with a second molecule, and the second molecule binds to the first molecule modified by the probe.

[0071] According to an embodiment of the present invention, the first molecule includes at least one of biotin, globin, cyclic dimer, SNAP-tag, CLIP-tag, or transcription factor tag, and the second molecule includes at least one of streptavidin, Halo Tag conjugate, FIAsH / ReAsH dye, dye corresponding to SNAP-tag, dye corresponding to CLIP-tag, or recognition sequence corresponding to transcription factor tag.

[0072] According to an embodiment of the present invention, the solid-phase carrier includes at least one of magnetic beads, silica gel, affinity resin, ion exchange resin, and hydrophilic gel.

[0073] According to an embodiment of the present invention, it further includes: subjecting the solid-phase carrier connected with the hybridization treatment product to enzymatic digestion treatment to obtain the cDNA of the TCR beta chain.

[0074] According to an embodiment of the present invention, it further includes: sequentially performing PCR amplification treatment on the cDNA of the TCR beta chain; performing library construction and sequencing on the PCR amplification treatment product.

[0075] Spatio-temporal transcriptome sequencing method

[0076] In the fourth aspect of the present invention, the present invention proposes a spatio-temporal transcriptome sequencing method. According to an embodiment of the present invention, the method includes: sequencing the TCR in a tissue sample to be tested by using the aforementioned method; and obtaining spatio-temporal transcriptome information of the tissue sample to be tested based on the sequencing result. The method of the present invention can improve the UMI capture number of the TCR beta chain, and can also improve the abundance of the TCR beta chain, which helps to locate more TCR-Ts that could not be detected in the past, obtain high-abundance full-length TCR sequence information, and solve the problem that the spatial position information of TCR cannot be captured in large quantities in the spatio-temporal transcriptome; in addition, the starting sample processed by this method is the cDNA of the stereo-seq library after sectioning and reverse transcription, not directly tissue RNA, the requirements for the conditions of the starting sample are reduced, and the experimental process has stability.

[0077] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0078] Example 1: Design of TCR beta chain probe

[0079] The specific experimental steps are as follows:

[0080] Design and synthesize 5-6 RNA probes targeting the TRBC gene sequence in the constant region of the TCR beta chain, and modify the 5' end of the RNA probes. The TCR multi-probe sequences used in the embodiments of the present invention are shown as SEQ ID NO:1-12 in Table 1 (where the TCR probe sequences shown as SEQ ID NO:1-9 are the shared sequences of the TRBC1 and TRBC2 genes).

[0081] Table 1

[0082]

[0083] Example 2: Enrichment method, spatio-temporal library construction and sequencing method of TCR beta chain probes based on multi-probe hybridization strategy. The schematic diagram of the technical solution of the present invention is shown in Figure 2 , and the specific experimental steps are as follows:

[0084] I. Obtaining of cDNA library samples (cDNA samples)

[0085] Perform tissue sectioning and patching, tissue fixation, fluorescence staining and photography, tissue permeabilization, reverse transcription reaction, tissue removal, cDNA release and recovery, cDNA purification and amplification steps on OCT-embedded mouse lymph node samples, mouse colorectal cancer tumors, and human colorectal cancer tumor samples in sequence according to the STOmics gene expression standard process to obtain stereo-seq spatio-temporal cDNA library samples.

[0086] II. Pre-hybridization

[0087] (1) Detect the concentration of the cDNA library in the previous step, calculate the volume required for 1000 ng of the cDNA library, and concentrate / dilute 1000 ng of the cDNA library to a quantitative volume of 9 μL by a vacuum concentrator / Nuclease-free water (Thermo) and transfer it to a PCR tube. Among them, the types and input amounts of the cDNA samples and probes in the PCR tube are specifically shown in Table 2.

[0088] Table 2

[0089]

[0090] (2) Put the cDNA library in the previous step into a PCR instrument and perform pre-hybridization according to the reaction conditions shown in Table 3.

[0091] Table 3

[0092] Step Temperature Time 1 95℃ 5 min 2 65℃ Wait

[0093] III. Hybridization

[0094] (1) In a new 0.2 mL PCR tube, use the reagent components in box 2 of the MGIEasy Exome Capture Elution Kit to prepare a single hybridization Buffer reaction system according to Table 4:

[0095] Table 4

[0096] Component Recommended volume (μL) AZCOHyb#1 (BGI) 10 AZCOHyb#2 (BGI) 0.4 AZCOHyb#3 (BGI) 4 AZCOHyb#4 (BGI) 5.6 Total volume 20

[0097] (2) Incubate the hybridization buffer in a PCR instrument at 65 °C for at least 5 min. Observe through a light source. It can be used only when there is no crystal precipitation in the system. Keep it at 65 °C for standby.

[0098] (3) Place the RNA probe stock solution on ice to melt. Dilute each RNA to 30 μM, 5 μL, and mix the TRBC probes of the same species in the same PCR tube. Pipette gently with a sterile tip and label it as probe mix.

[0099] (4) Take 1 μL of probe mix and use a UV spectrophotometer to detect the RNA concentration. Calculate the volume and dilute it quantitatively to 5 μL, 220 ng / μL probe mix in a new PCR tube according to the actual detected concentration. Label it as Probemix(220 ng / μL).

[0100] (5) Add the reagent component RNase Block (Block5) in box 1 of the MGIEasy Exome Capture Elution Kit to the Probe mix(220 ng / μL) tube according to the required amount for a single hybridization system in Table 5 to prepare the following probe mixture system. Then cover the PCR tube lid:

[0101] Table 5

[0102] Reagent Volume (μL) Nuclease-free water (Thermo) 2.75 Probe mix (220 ng / μL) 3.75 RNase Block (Block5) 0.5 Total volume 7

[0103] (6) Put the probe mixture into a PCR instrument and incubate it at 65 °C for 2 min and wait.

[0104] (7) Keep each reaction system at 65 °C. Open the lids of the sample library tube and the hybridization Buffer tube, quickly transfer 13 μL of the hybridization buffer to the sample library tube, and then cover the PCR tube lid.

[0105] (8) Keep the PCR tube at 65 °C (set the hot lid of the thermal cycler to 105 °C) for hybridization for more than 24 h.

[0106] (9) After 24 hours of hybridization, prepare the MGIEasy Exome Capture Elution Kit box2 and Dynabeads M-280 Streptavidin Beads (Thermo). Set the thermostat to 65 °C at least 30 minutes in advance. Take 1.8 mL of WashBuffer II (MGIEasy Exome Capture Elution Kit box2) into a 2.0 mL centrifuge tube and preheat it to 65 °C in the thermostat.

[0107] IV. TCR Single-strand Elution

[0108] (1) Vigorously resuspend the Dynabeads M-280 Streptavidin Beads with a vortex mixer until well mixed. Take 50 μL of Dynabeads M-280 Streptavidin Beads into a new 1.5 mL centrifuge tube.

[0109] (2) Take 200 μL of Binding buffer (MGIEasy Exome Capture Elution Kit box2) and add it to the magnetic beads. Vigorously shake the magnetic beads with a vortex mixer for 5 seconds to resuspend them.

[0110] (3) Place the centrifuge tube on the magnetic rack for 2 minutes until the liquid is completely clear; carefully aspirate and discard the supernatant.

[0111] (4) Repeat the previous two steps twice.

[0112] (5) Add 200 μL of Binding buffer to resuspend the magnetic beads.

[0113] (6) After 24 hours of incubation, keep the hybridization mixture on the PCR instrument and set the temperature to 60 °C and wait. Quickly measure the volume of the remaining hybridization mixture in the PCR tube with a pipette.

[0114] (7) Directly transfer all the hybridization mixture (about 25 μL) from the PCR instrument to the prepared magnetic beads. Invert the centrifuge tube 3 to 5 times until well mixed.

[0115] (8) Symmetrically fix the centrifuge tube containing the hybridization mixture and magnetic beads on the rotator and rotate it 360 degrees to mix well. Incubate at room temperature for 30 minutes.

[0116] (9) After the magnetic bead capture incubation, perform magnetic bead elution to obtain the magnetic bead capture product:

[0117] ① Remove the centrifuge tube from the mixing device and centrifuge briefly to ensure that there is no liquid residue on the tube cap.

[0118] ② Transfer the centrifuge tube to the magnetic rack and let it stand for 3 - 5 minutes until the liquid is completely clear. Carefully aspirate and discard the supernatant.

[0119] ③ Resuspend the magnetic beads with 500 μL of Wash Buffer I (from box 2 of the MGIEasy Exome Capture Elution Kit), invert the tube up and down until the magnetic beads are completely mixed, and incubate the sample at room temperature for 15 min.

[0120] ④ Centrifuge the centrifuge tube briefly, then place it on the magnetic stand, let it stand for 3 - 5 min until the liquid is completely clear, and carefully aspirate and discard the supernatant.

[0121] ⑤ Resuspend the magnetic beads with 500 μL of pre - heated Wash Buffer II (from box 2 of the MGIEasy Exome Capture Elution Kit), and vortex for 5 s to mix the sample, then incubate it in a thermostatic instrument at 65 °C for 10 min.

[0122] ⑥ Invert the centrifuge tube to mix the sample, centrifuge briefly for 3 s, then place it on the magnetic stand, let it stand for 5 min until the liquid is completely clear, and carefully aspirate and discard the supernatant.

[0123] ⑦ Repeat the previous two steps twice.

[0124] ⑧ Resuspend the magnetic beads with 85 μL of NF - water (Thermo), and use a pipette to aspirate all the resuspended magnetic bead - captured products (including the magnetic beads) into a new PCR tube.

[0125] (10) Unwind the magnetic bead products to release TCR products:

[0126] ① Take out the RNaseH enzyme reagent and let it melt on ice.

[0127] ② Prepare the following enzymatic digestion system 1 as shown in Table 6 in the PCR tube of the magnetic bead - captured product:

[0128] Table 6

[0129]

[0130] ③ Put it into a PCR instrument and incubate at 37 °C for 20 min.

[0131] ④ Place the reacted centrifuge tube on the magnetic stand for 2 min, wait until the liquid is completely clear, carefully aspirate the supernatant (TCRssDNA) into a new EP tube (about 90 - 100 μL), and add 1 μL of 0.5 M EDTA to the supernatant to terminate the enzymatic digestion.

[0132] ⑤ Add 50 μL of enzymatic digestion system 2 to the magnetic beads on the magnetic stand, put it into a PCR instrument, and incubate at 37 °C for 20 min.

[0133] ⑥ Place the centrifuge tube after the reaction on the magnetic rack for 2 minutes. When the liquid is completely clear, carefully pipette the supernatant (TCRssDNA) into a new EP tube (about 40-50 μL), and add 0.5 μL of 0.5M EDTA to the supernatant to terminate the enzymatic hydrolysis.

[0134] (11) The enzymatic product was purified using 0.8x DNA clean beads (Vazyme):

[0135] ① Take out the DNA Clean Beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use.

[0136] ②Measure the volume of the enzymatic hydrolysis product in the previous step, pipette 0.8x DNA Clean Beads into the enzymatic hydrolysis product, and shake to mix.

[0137] ③Incubate at room temperature for 10 minutes.

[0138] ④ Centrifuge the centrifuge tube instantaneously, place it on a magnetic rack, let it stand for 2-5 minutes until the liquid becomes clear, carefully aspirate the supernatant with a pipette and discard it.

[0139] ⑤ Keep the centrifuge tube on the magnetic rack, add 1 mL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate the supernatant and discard it.

[0140] ⑥ Repeat the previous step and try to dry the liquid in the tube. If there is a small amount of liquid left on the tube wall, centrifuge the tube instantly. After separation on the magnetic rack, use a small-range pipette to dry the liquid at the bottom of the tube.

[0141] ⑦ Keep the centrifuge tube on the magnetic rack, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0142] ⑧ Remove the centrifuge tube from the magnetic rack, add 40 μL NF-H2O to dissolve, shake and mix, let stand at room temperature for 5 minutes, centrifuge instantly, let stand on the magnetic rack for 3-5 minutes, and recover the supernatant TCR product after the liquid is clarified.

[0143] ⑨Take 1 μL of the sieve product and use Qubit dsDNA HS AssayKit to detect the concentration.

[0144] 5. Amplification

[0145] (1) PCR amplification of enriched products:

[0146] ① Calculate the volume required to take 100 ng of the TCR product according to the concentration of the TCR product detected in Step 4 of this embodiment, prepare the PCR Mix, vortex 3 times, 3 s each time, and centrifuge instantaneously to collect the reaction solution at the bottom of the tube. Among them, the PCR Mix is shown in Table 7-1, and the specific value of the cDNA volume X in the embodiment is shown in Table 7-2. Table 7-1

[0147] Reagent Volume (μL) 1X cDNA (TCR ssDNA: 100 ng) X cDNA amplification Mix (BGI) 50 cDNA Primers (BGI) 8 Nuclease-free water (Thermo) Up to 100 μL Total 100

[0148] Table 7-2

[0149] Experimental group cDNA volume (μL) 1X 1 32 2 30 3 28 4 36 5 34 6 38 7 38

[0150] ② Perform PCR according to the following procedure as shown in Table 8:

[0151] Table 8

[0152]

[0153]

[0154] ③ After the reaction is completed, centrifuge instantaneously to collect the reaction solution at the bottom of the tube.

[0155] ④ Use a pipette to aspirate 100 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube.

[0156] (2) Purification after enrichment product PCR:

[0157] ① Take out 0.8x DNA Clean Beads 30 min in advance and place them at room temperature. Vortex well before use.

[0158] ② Aspirate 80 μL of DNA Clean Beads into 100 μL of the post-hybridization PCR product and vortex well.

[0159] ③ Incubate at room temperature for 10 min.

[0160] ④ Centrifuge the centrifuge tube instantaneously, place it on the magnetic rack, let it stand for 2 - 5 min until the liquid is clear, and carefully aspirate and discard the supernatant with a pipette.

[0161] ⑤ Keep the centrifuge tube on the magnetic rack, add 1 mL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall, let it stand for 30 s, and then carefully aspirate and discard the supernatant.

[0162] ⑥ Repeat Step ⑤, try to dry the liquid in the tube as much as possible. When there is a small amount of liquid remaining on the tube wall, centrifuge the centrifuge tube instantaneously, separate it on the magnetic rack, and then use a pipette with a small range to dry the liquid at the bottom of the tube.

[0163] ⑦ Keep the centrifuge tube on the magnetic rack, open the tube cap of the centrifuge tube, and dry it at room temperature until there is no reflection and no cracking on the surface of the magnetic beads.

[0164] ⑧ Remove the centrifuge tube from the magnetic stand, add 50 μL of NF-H2O for re-dissolution, mix well by shaking, let stand at room temperature for 5 min, centrifuge instantaneously, and let stand on the magnetic stand for 3 - 5 min. After the liquid becomes clear, recover the supernatant product.

[0165] (3) Identification of the enriched product:

[0166] ① Take 1 μL of the product after chip screening and detect the concentration using the Qubit dsDNA HS Assay Kit.

[0167] ② Dilute the sample to a concentration of 1 - 2 ng / μL and a volume of 15 - 20 μL using the 1X dilution buffer of the qseq100 kit (Guangding Biotech) according to the detected concentration, and perform fragment distribution detection using the qseq100 kit, and compare with the fragment lengths of the sample before enrichment. The product fragments should meet the requirements of the spatial-temporal library fragments before enrichment, approximately 600 - 1500 bp.

[0168] VI. Sequencing

[0169] Performing spatial-temporal library construction and sequencing on the enriched product can increase the number of captured reads of the TCR beta chain. Sending the enriched product for single-molecule sequencing can obtain the full-length sequence information of the TCR beta chain.

[0170] VII. Experimental Results

[0171] Perform experiments on experimental groups 1 - 7 through the above steps, and the results of the specific number of sequencing reads are shown in Table 9 below:

[0172] Table 9

[0173]

[0174]

[0175] The results show that the present invention can effectively increase the proportion of the sequencing capture rate of the TCR beta chain in the stereo-seq cDNA sample. This embodiment exemplarily shows the experimental results (experimental groups 1 - 5) of enriching mouse lymph node samples and mouse colorectal cancer tumors using a mixed probe consisting of Mouse-TRBC-v1 - v4 (SEQ ID NO: 6 - 9), Mouse-TRBC1-v5 (SEQ ID NO: 10); the experimental results (experimental group 6) of enriching human colorectal cancer tumor samples using a mixed probe consisting of Human-TRBC-v1 - v5 (SEQ ID NO: 1 - 5), and the experimental results (experimental group 7) of enriching human colorectal cancer tumor samples using a single probe of Human-reference change-RNA (SEQ ID NO: 12).

[0176] In mouse lymph node samples, the multi-probe strategy of the present invention (Mouse-TRBC-v1 to v4 (SEQ ID NO: 6 to 9), Mouse-TRBC1-v5 (SEQ ID NO: 10)) increased the TRBC / total UMI counts ratio to approximately 1000-fold (750 - 2200), and the detectable TRBC UMI count increased by 18 - 78 times; in mouse colorectal cancer tumor samples, the multi-probe strategy of the present invention (Mouse-TRBC-v1 to v4 (SEQ ID NO: 6 to 9), Mouse-TRBC1-v5 (SEQ ID NO: 10)) increased the TRBC / total UMI counts ratio to 5016-fold, and the detectable TRBC UMI count increased by 18 times. In human colorectal cancer samples, the multi-probe strategy of the present invention (Human-TRBC-v1 to v5 (SEQ ID NO: 1 to 5)) increased the TRBC / total UMI counts ratio by 1693-fold, and the detectable TRBC UMI count increased by 77 times. Compared with the single-probe hybridization capture of the Human-reference change-RNA probe shown in SEQ ID NO: 12, the TRBC / total UMI counts ratio of the single-probe hybridization only increased by 3.5-fold, and the detectable TRBC UMI count did not increase.

[0177] Secondly, spatio-temporal clustering analysis showed that the expression levels of TRBC in mouse tissue samples and human colorectal cancer tissue samples were significantly increased before and after enrichment, and the spatial positions were consistent. Through the analysis of Tfh-specific gene expression and clustering, it was shown that the enriched product could capture the TCR beta copies in the region where Tfh was located, and the TCR beta chain at this spatial position had a low expression level before enrichment and could not be effectively captured by sequencing.

[0178] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A probe set, characterized in that, Comprising one or more probes; wherein, the probe is RNA, the probe is capable of complementary base pairing with at least a partial sequence of the constant region of the TCR beta chain.

2. The probe set according to claim 1, wherein The probe is capable of complementary base pairing with at least a partial sequence of the TRBC gene of the constant region of the TCR beta chain; Optionally, the TRBC gene of the constant region of the TCR beta chain is derived from human or murine; Optionally, the probe comprises a nucleotide sequence as shown in any one of SEQ ID NO: 1-11; Optionally, the probe set comprises at least two probes; Optionally, the probe set comprises a nucleotide sequence as shown in SEQ ID NO: 1-5; Optionally, the probe set comprises a nucleotide sequence as shown in SEQ ID NO: 6-9, and a nucleotide sequence as shown in SEQ ID NO: 10 and / or SEQ ID NO: 11; Optionally, the 5'-end of the probe has a first molecular modification; Optionally, the first molecule comprises at least one of biotin, globin, cyclic dimer, SNAP-tag, CLIP-tag or transcription factor tag.

3. A kit, characterized in that, Comprising: The probe set according to any one of claims 1-2.

4. A method for enriching TCR beta chain, characterized in that, Comprising: Hybridizing the probe set according to any one of claims 1-2 or the kit according to claim 3 with a sample to be tested, wherein the sample to be tested contains the TCR beta chain; Collecting the hybridization product to obtain the TCR beta chain.

5. The method according to claim 4, wherein The collecting of the hybridization product is carried out by the following steps: Contacting a solid-phase carrier with the hybridization product to obtain a solid-phase carrier connected with the hybridization product; wherein, the solid-phase carrier is connected with a second molecule, and the second molecule binds to the first molecule; Optionally, the first molecule comprises at least one of biotin, globin, cyclic dimer, SNAP-tag, CLIP-tag and transcription factor tag, and the second molecule comprises at least one of streptavidin, Halo Tag conjugate, FIAsH / ReAsH dye, dye corresponding to SNAP-tag, dye corresponding to CLIP-tag and recognition sequence corresponding to transcription factor tag; Optionally, the solid-phase carrier comprises at least one of magnetic beads, silica gel, affinity resin, ion exchange resin and hydrophilic gel; Optionally, further comprising: Performing enzymatic digestion on the solid-phase carrier connected with the hybridization product to obtain the TCR beta chain.

6. The method according to claim 5, wherein the sample to be tested is a cDNA sample; Optionally, the cDNA sample is obtained by performing RNA capture and reverse transcription on a tissue sample to be tested.

7. A method for TCR sequencing, characterized in that, Comprising: Hybridizing the sample to be tested with the probe set according to any one of claims 1-2 or the kit according to claim 3, wherein the sample to be tested contains the TCR beta chain; Collecting the hybridization product to obtain the TCR beta chain; Constructing a library and performing sequencing on the TCR beta chain.

8. The method according to claim 7, wherein The TCR beta chain includes the nucleotide sequence of the full-length TCR beta chain; Optionally, the nucleotide sequence is a cDNA sequence.

9. The method according to claim 8, wherein The sample to be tested is a cDNA sample; Optionally, the cDNA sample is obtained by performing RNA capture and reverse transcription on the tissue sample to be tested.

10. The method according to claim 9, characterized in that The collection of the hybridization treatment product is carried out by the following steps: Contact the solid-phase carrier with the hybridization treatment product to obtain a solid-phase carrier connected with the hybridization treatment product; Wherein, the solid-phase carrier is connected with a second molecule, and the second molecule binds to the first molecule modified by the probe; Optionally, the first molecule includes at least one of biotin, globin, cyclic dimer, SNAP-tag, CLIP-tag, or transcription factor tag, and the second molecule includes at least one of streptavidin, Halo Tag conjugate, FIAsH / ReAsH dye, dye corresponding to SNAP-tag, dye corresponding to CLIP-tag, or recognition sequence corresponding to the transcription factor tag; Optionally, the solid-phase carrier includes at least one of magnetic beads, silica gel, affinity resin, ion exchange resin, and hydrogel; Optionally, further includes: Perform enzymatic digestion on the solid-phase carrier connected with the hybridization treatment product to obtain the cDNA of the TCR beta chain; Optionally, further includes: Perform PCR amplification on the cDNA of the TCR beta chain in sequence; Perform library construction and sequencing on the PCR amplification product.

11. A TCR beta-chain spatiotemporal transcriptome sequencing method, characterized in that, Includes: Sequencing the TCR in the tissue sample to be tested by using the method according to any one of claims 7 to 10; And Based on the sequencing results, obtain the spatiotemporal transcriptome information of the TCR beta chain of the tissue sample to be tested.

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