Method for simultaneously detecting TCR and HLA genotyping and application

文档序号：336628 发布日期：2021-12-03 浏览：40次中文

阅读说明：本技术 同时检测tcr和hla基因分型的方法及应用 (Method for simultaneously detecting TCR and HLA genotyping and application ) 是由杨凡林莉娅张伟于 2021-08-11 设计创作，主要内容包括：本发明涉及一种同时检测TCR和HLA基因分型的方法及应用,属于基因检测技术领域。该方法包括以下步骤：样品提取：取待测生物样本,提取其中DNA；PCR扩增：取上述DNA,同时加入TCR扩增引物和HLA扩增引物,对TCR目标区域和HLA目标区域进行同时扩增,所述HLA目标区域为HLA基因的2,3,4号外显子区域；文库构建：取上述扩增产物,构建文库；上机测序：取上述文库,上机进行高通量测序；数据分析：对上述测序数据进行处理分析,获得TCR和HLA基因分型情况。采用上述方法可在不增加建库步骤的情况下,在同一PCR反应体系内的HLA引物组扩增HLA-I型基因进行分型鉴定,获取TRB信息的同时提供HLA的分型结果,从而获取免疫组库信息评估样本提供者的健康状态。(The invention relates to a method for simultaneously detecting TCR and HLA genotyping and application thereof, belonging to the technical field of gene detection. The method comprises the following steps: sample extraction: taking a biological sample to be detected, and extracting DNA in the biological sample; and (3) PCR amplification: taking the DNA, simultaneously adding a TCR amplification primer and an HLA amplification primer, and simultaneously amplifying a TCR target region and an HLA target region, wherein the HLA target region is an exon region 2,3 or 4 of an HLA gene; library construction: taking the amplification product to construct a library; and (3) machine sequencing: taking the library, and performing high-throughput sequencing on the library; and (3) data analysis: and processing and analyzing the sequencing data to obtain the TCR and HLA genotyping conditions. By adopting the method, the HLA primer group in the same PCR reaction system can amplify HLA-I type genes for typing identification without increasing the step of establishing the library, and the typing result of HLA is provided while TRB information is obtained, so that the health state of the immune repertoire information evaluation sample provider is obtained.)

1. A method for simultaneously detecting TCR and HLA genotyping, comprising the steps of:

sample extraction: taking a biological sample to be detected, and extracting DNA in the biological sample;

and (3) PCR amplification: taking the DNA, simultaneously adding a TCR amplification primer and an HLA amplification primer, and simultaneously amplifying a TCR target region and an HLA target region, wherein the HLA target region is an exon region 2,3 or 4 of an HLA gene;

library construction: taking the amplification product to construct a library;

and (3) machine sequencing: taking the library, and performing high-throughput sequencing on the library;

and (3) data analysis: and processing and analyzing the sequencing data to obtain the TCR and HLA genotyping conditions.

2. A method of simultaneously detecting both TCR and HLA genotyping according to claim 1 wherein in the PCR amplification step, the TCR target regions are the V region and the J region of CDR3 in the TRB; the HLA target region is HLA-A gene exon 2-4, HLA-B gene exon 2-4 and HLA-C gene exon 2-4, and the target product segment length is 200-500 bp.

3. A method for simultaneously detecting genotyping of both TCR and HLA as claimed in claim 2 wherein in the PCR amplification step, the TCR amplification primers comprise the sequences set forth in SEQ ID No.1-SEQ ID No. 41; the HLA amplification primer comprises a sequence shown as SEQ ID NO.42-SEQ ID NO. 60.

4. A method for simultaneously detecting genotyping of TCR and HLA as claimed in claim 3 wherein in the PCR amplification step, the TCR amplification primers are used in the following ratios:

the HLA amplification primers are used according to the following proportion:

5. a method for simultaneously detecting genotyping of both TCR and HLA as claimed in claim 1 wherein in the PCR amplification step, the total molar ratio of TCR amplification primers to HLA amplification primers is 8-12: 1.

6. A method for simultaneously detecting genotyping of both TCR and HLA as claimed in claim 1 wherein in the PCR amplification step, the total concentration of said TCR and HLA amplification primers is from 0.3 to 0.5 mM.

7. Use of a method of simultaneous detection of TCR and HLA genotyping according to any one of claims 1 to 6 in the preparation of reagents and/or devices for TCR and HLA genotyping.

8. A kit for simultaneously detecting TCR and HLA genotyping is characterized by comprising a TCR amplification primer and an HLA amplification primer which simultaneously amplify a TCR target region and an HLA target region.

9. The kit of claim 8, wherein the TCR target region is the V and J regions of CDR3 in the TRB; the HLA target region is HLA-A gene exon 2-4, HLA-B gene exon 2-4 and HLA-C gene exon 2-4.

10. A system for simultaneously detecting TCR and HLA genotyping, comprising:

a detection device for detecting according to the method of any one of claims 1 to 6 to obtain sequencing data;

the analysis device is used for acquiring the sequencing data to analyze and process so as to obtain the TCR and HLA genotyping conditions;

and the output device is used for outputting the TCR and HLA genotyping conditions.

Technical Field

The invention relates to the technical field of gene detection, in particular to a method for simultaneously detecting TCR and HLA genotyping and application thereof.

Background

T lymphocyte surface receptors (TCRs) are heterodimers of molecular structures that specifically recognize and bind antigen peptide-MHC molecules by T cells, and are composed of two distinct subunits. 95% -99% of the T cell receptors are composed of alpha and beta subunits, and 1% -5% of the T cell receptors are composed of gamma and delta subunits, the ratio varying with the development of the individual and the health of the body.

TRB (T Cell Receptor β Locus) is a Locus that encodes the β peptide chain in the TCR molecule. The CDR3 of TRB is composed of four gene regions, V (variable), D (conversion), J (joining) and C (constant), each of which contains several alleles. Gene rearrangement in the lymphocyte maturation process forms various recombinant sequence segments, and the insertion and mutation of DNA base forms T cell diversity. The beta chain in the TCR takes precedence over the alpha chain of the TCR in an allelic exclusion way, and the characteristics of the T cell TCR can be better reflected. Therefore, the method has very important significance for the research of TRB diversity. The α and β peptide chains of the TCR are each composed of, inter alia, a Variable region (Variable), a constant region (Diversity) transmembrane region and a cytoplasmic region. The variable Region consists of three Complementarity Determining Regions (CDRs) CDR1, CDR2, CDR3, of which CDR3 is the most variable and directly determines the antigen binding specificity of the TCR.

An Immune Repertoire (Immune repterore) sequencing technology taking a T/B lymphocyte surface receptor as a research target can deeply evaluate the Immune state of an organism and explore the relationship between the Immune state of a human body and diseases. With the development of high-throughput sequencing, large-scale sequencing analysis such as immune repertoire and the like is realized. By sequencing the CDR3 region of TRB in T cells, parameters such as TRB diversity associated with human health can be obtained.

In addition, the human leukocyte antigen system HLA (HLA) gene is located in the short arm of human chromosome 6, which is the most diverse and important immune genetic system in the human genome, and is the main genetic system for regulating the specific immune response of human body and determining the individual difference of disease susceptibility. The diversity of HLA determines the body's resistance to infectious diseases, autoimmune diseases, genetic diseases and various tumors. The HLA system plays an important role in antigen recognition, antigen presentation, immune response and regulation, destruction of foreign antigen target cells and the like, and is the main material basis for causing immunological rejection.

HLA genes are classified into three classes: class I, class II and class III genes, wherein class I includes: HLA-A, HLA-B, HLA-C genes. Both class I and class II antigens on the cell surface of the graft are strong graft antigens, and both humoral and cellular immunity are involved in rejection of the graft, whether xenogenic organ or tissue or cell transplantation, for HLA matching between recipients is critical to success. With the development of medicine, new genetic technologies can be used for typing detection, such as leukemia and thalassemia, and then suitable donors can be searched for transplantation treatment.

At present, the high-resolution typing of HLA is adopted, the matching effect can be greatly improved by the peripheral blood stem cell transplantation technology, the higher the matching degree of HLA-related genes of a donor and a receptor is, the higher the differentiation rate is, the higher the transplantation success rate can be, and the faster and more secure the rehabilitation of patients can be realized.

In addition, HLA typing can also assist in diagnosing certain diseases and preventing serious adverse drug reactions. Over 200 diseases have been found to occur in association with HLA, including some autoimmune and infectious diseases, and more research has been directed to HLA and cancer susceptibility.

The diversity of HLA has close relation with the resistance of organisms to infectious diseases, autoimmune diseases, genetic diseases and various tumors, so HLA typing information can be used for auxiliary diagnosis of diseases and prevention of serious adverse drug reactions, and becomes a basic reference basis for disease treatment and traceability.

HLA typing methods include serotyping, cytological typing and molecular biological typing. With the development of molecular biology and DNA sequencing technologies, traditional serological and cytological typing methods have been gradually replaced by molecular biology methods. Currently, the main methods for typing HLA molecules are: polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction-sequence specific primer (PCR-SSP), polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP), polymerase chain reaction oligonucleotide probe hybridization (PCR-SSO), sequencing-based typing (PCR-SBT), and the like.

The serology method mainly comprises the steps of analyzing the HLA type of a subject through an HLA trace lymphocyte toxicity test method, separating lymphocytes from whole blood or lymph tissues, incubating the lymphocytes with a micropore plate coated with an HLA-specific antibody, adding complement, and enabling the specific antibody capable of being combined with HLA antigens on the surfaces of the lymphocytes to activate the complement through a classical pathway, so that target cells are hydrolyzed (lymphocyte toxicity). There are many limitations to this approach, for example, in determining HLA-specific antibodies requires extensive screening work, some HLA antigens are difficult to identify by serological methods, HLA cross-reactivity affects the accuracy of the typing results, and difficulties arise in further subtype determinations.

The cytological typing method is mainly detected by experiments of homozygous typing cells and sensitized lymphocytes, and the basic principle of the cosmetic method is to judge the proliferation reaction of the lymphocytes after recognizing the non-self HLA antigenic determinant. The method is gradually eliminated due to the difficult sources of the parting cells and the complicated operation means.

PCR-FRLP (restriction fragment length polymorphism) refers to the process of cutting DNA into fragments with different sizes under the action of restriction enzyme, utilizing the difference of electrophoretic mobility, and analyzing target genes by electrophoretic separation, blotting, probe hybridization and other technologies.

PCR-SSP (Sequence-specific primer) is a set of allele-specific primers designed, and DNA fragments obtained by PCR amplification are distinguished by different molecular weights in the process under electrophoresis.

In PCR-SSCP (single strand transformation for polymorphism), PCR products are denatured into single DNA strands, separated by gel electrophoresis, only one nucleotide is mutated, and the electrophoretic migration is dry-fried, so that the PCR products can be used for polymorphism analysis.

PCR-SSO (sequence specific oligonucleotide) uses an artificially synthesized HLA type-specific oligonucleotide sequence as a probe to hybridize with the HLA gene fragment amplified by PCR, thereby determining the HLA type.

PCR-SBT (sequence based typing) is a target sequence of HLA genes obtained by PCR, and HLA type results are obtained by sequencing analysis, and the method is widely used along with the development of NGS and is also the 'gold standard' recommended by the world health organization at present.

In the technical scheme, the serum method and the cell method are fussy to operate and are gradually eliminated, PCR-FRLP, PCR-SSP and PCR-SSCP take gel electrophoresis separation experiment results as typing according to the process, great uncertainty is brought, and great influence is brought to HLA typing accuracy. Therefore, the SBT method needs further optimization of HLA primers to obtain accurate typing results through second generation sequencing analysis.

In addition, the conventional library construction scheme is to separate TRB and HLA for library construction, only one of sequencing results can be analyzed, and when HLA is needed to assist diagnosis, one step of library construction, sequencing and analysis is needed, which results in waste of time and increase of cost.

Disclosure of Invention

Therefore, there is a need to provide a method for simultaneously detecting TCR and HLA genotyping, which can obtain TCR diversity and HLA information at one time, combine HLA typing results and TCR immune status, facilitate comprehensive evaluation, and provide more accurate and targeted key information for subsequent adjuvant therapy.

A method for simultaneously detecting TCR and HLA genotyping, comprising the steps of:

sample extraction: taking a biological sample to be detected, and extracting DNA in the biological sample;

library construction: taking the amplification product to construct a library;

and (3) machine sequencing: taking the library, and performing high-throughput sequencing on the library;

and (3) data analysis: and processing and analyzing the sequencing data to obtain the TCR and HLA genotyping conditions.

The inventor finds in previous research that although conventional technologies have a scheme for detecting a TRB-only and an HLA-only library, it is difficult to library the TRB and the HLA in the same reaction, and problems such as interaction between multiple primers and coverage of the HLA on A, B, C three genes are encountered.

In HLA typing research, the full length of HLA is about 3.9kb, and the reference value of conservative intron without typing is mainly based on the research of abundant diversified exons. The total number of HLA-I exons is 7, and the 2,3 and 4 exons with highest diversity are mainly used as the major part in the typing process, so that the 2,3 and 4 exons as the typing genes have more accurate reference value.

On the basis, the inventor designs the amplification primers of the HLA genes to only aim at the 2,3 and 4 exon regions with the most abundant diversity by repeatedly searching and trying, reduces the target regions of the HLA genes under the condition of not influencing HLA typing, shortens the amplification length of the HLA exons, and simultaneously obtains the HLA type information in the sequencing of the immune repertoire TRB library.

In one embodiment, in the PCR amplification step, the TCR target region is the V region and J region of CDR3 in the TRB;

the HLA target region is HLA-A gene exon 2-4, HLA-B gene exon 2-4 and HLA-C gene exon 2-4, and the target product segment length is 200-500 bp.

By selecting a specific TCR target region and an HLA target region and controlling the size of a target product of an HLA gene to be 200bp-500bp (preferably 296bp-360bp), the multiplex PCR amplification effect is further improved, and a foundation is laid for high-quality library establishment in the follow-up process.

In one embodiment, in the PCR amplification step, the TCR amplification primer comprises a sequence shown in SEQ ID No.1-SEQ ID No. 41; the HLA amplification primer comprises a sequence shown as SEQ ID NO.42-SEQ ID NO. 60.

The primer design of the present invention must include amplification of almost all HLA types to be suitable for HLA type validation of different people; the primer binding site needs to avoid a non-conservative region, and a proper specific primer site is searched near the upstream and downstream of the A, B, C exon as much as possible; due to the read length design of PE150, there should be less polymorphism in the gap region that fails to obtain sequence by sequencing in amplifying products larger than 300bp to improve the resolution of HLA typing. On the basis, the primer sequence can achieve better typing resolution.

In one embodiment, in the PCR amplification step, the TCR amplification primers are used in the following ratios:

the HLA amplification primers are used according to the following proportion:

considering that differences among exons, caused by differences among sample individuals and abundance of HLA-I type exons, and problems that amplification coverage is small in multiplex PCR and the like possibly occur, and data needed by subsequent typing is insufficient or distorted, the primer proportion is adjusted through repeated experiments, and effective data of each HLA exon through adjustment of the primer proportion reach the data amount capable of being used for typing; the problems are successfully solved, no mutual influence exists between the TRB library establishing primer and the HLA library establishing primer through tests, the data volume of A, B, C genes is properly distributed in the HLA library establishing process, and wider coverage and establishment of a diversity amplification system are realized.

In one embodiment, in the PCR amplification step, the total molar ratio of TCR amplification primers to HLA amplification primers is 8-12: 1.

In order to enable the TRB primer group to obtain data volume which is higher than HLA and achieve HLA typing data volume in the same DNA sequencing library to achieve better typing effect, the proportion of the two groups of primers can be properly adjusted, and finally, the proportion of 8-12:1 can be determined to obtain more ideal data proportion.

In one embodiment, the total concentration of TCR and HLA amplification primers in the PCR amplification step is 0.3-0.5 mM.

The invention also discloses application of the method for simultaneously detecting TCR and HLA genotyping in preparing reagents and/or equipment for TCR and HLA genotyping.

The invention also discloses a kit for simultaneously detecting the TCR and HLA genotyping, which comprises a TCR amplification primer and an HLA amplification primer for simultaneously amplifying the TCR target region and the HLA target region.

In one embodiment, the TCR target region is the V and J regions of CDR3 in the TRB; the HLA target region is HLA-A gene exon 2-4, HLA-B gene exon 2-4 and HLA-C gene exon 2-4.

The invention also discloses a system for simultaneously detecting TCR and HLA genotyping, which comprises:

the detection device is used for detecting according to the method to obtain sequencing data;

the analysis device is used for acquiring the sequencing data to analyze and process so as to obtain the TCR and HLA genotyping conditions;

and the output device is used for outputting the TCR and HLA genotyping conditions.

Compared with the prior art, the invention has the following beneficial effects:

according to the method for simultaneously detecting the genotyping of the TCR and the HLA, the HLA-I type gene is amplified through the HLA primer group in the same PCR reaction system for typing identification under the condition of not increasing a library building step, the HLA typing result is provided while TRB information is obtained, and the method can be used as information reference of disease traceability and organ transplantation and the like so as to obtain immune library information.

By using the method, the diversity of the TCR and the HLA information can be acquired at one time, the typing cost of the HLA is reduced, and the disease source in the sample is screened in combination with the HLA typing result and the TCR immune state, so that the immunodetection evaluation can be better performed, and more accurate and in-place key information is provided for the subsequent adjuvant therapy.

In addition, the invention obtains two sets of multiplex PCR primer groups which are not mutually influenced in respective target areas through design and experimental verification, can realize the amplification of various TRB and HLA genes in one reaction by adopting a multiplex PCR method, quickly enriches and establishes TRB and HLA libraries, analyzes the composition of TRB and the typing of HLA in a sample by using an NGS sequencing technology, and has important significance in revealing human health and disease analysis.

Drawings

FIG. 1 is a technical scheme of example 1;

FIG. 2 is a statistical chart of the effective data rate of A, B, C gene for HLA-I gene before the adjustment of the primer ratio of each exon in example 1;

FIG. 3 is a statistical chart of the effective data rate of the HLA-I type gene A, B, C obtained by adjusting the ratio of primers for each exon in the gene in example 1;

FIG. 4 is a graph showing the difference in preference before the proportion of TRB primers is adjusted in example 1;

FIG. 5 is a graph showing the difference in preference among the TRB primers in example 1 after the proportion thereof has been adjusted;

FIG. 6 is a gel electrophoresis image of HLA (upper) and TRB (lower) gene fragments obtained under the same multiplex PCR reaction in example 1;

FIG. 7 is a CDR3 length distribution diagram of the TRB gene in example 1;

FIG. 8 is a CDR3 abundance distribution map of the TRB gene in example 1;

FIG. 9 is a CDR3 histogram (left) Top10 CDR3 histogram (right) of example 1;

FIG. 10 is a three-dimensional V-J pairing map of the TRB gene in example 1.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the following examples, unless otherwise specified, are all routinely practiced.

Example 1

A method for simultaneously detecting TCR and HLA genotyping, the technical route flow is shown in figure 1, and comprises the following steps:

1. sample collection

Peripheral blood was extracted 5ml using an EDTA tube, and the tube was inverted several times to prevent coagulation.

2. Sample extraction

2ml of Blood was taken into an EP tube, and DNA of the sample was obtained using a Blood DNA extraction Kit (Hipure Blood DNAmidi Kit I, D312-02, magenta) and according to the instructions, and quantified using Q-bit for DNA library construction.

3. PCR amplification

Taking 1.2 mu g of the DNA, adding TCR amplification primers and HLA amplification primers according to the proportion shown in the following table 1, and capturing and enriching TCR VJ regions and No. 2,3 and 4 exon regions of HLA-A, B, C by using a multiplex PCR method.

In the early work, the primer input amount is adjusted and optimized in the step, BWA comparison is carried out on the following machine data, and the proportion of each internal standard sequence primer before and after adjustment is determined. FIGS. 2 to 3 are examples of TRB and HLA amplification distribution before the adjustment of the primer ratio, respectively, and FIGS. 4 to 5 are examples of TRB and HLA amplification distribution after the adjustment of the primer ratio, respectively.

The amplification primers are equivalently added according to a mode that the molar ratio is 1, a more serious amplification deviation exists in the multiplex PCR process, as shown in FIG. 2, the abscissa in the figure is respectively different samples and different genes (the same depth is the same sample, namely, each group of data is samples 1-4 from left to right), the ordinate is the effective data proportion after each gene is amplified, the abscissa in FIG. 3 is respectively different samples and different genes, and the ordinate is the effective data proportion after each gene is amplified, as can be seen from FIGS. 2-3, a more serious amplification deviation exists according to the mode of equivalent addition.

By using the internal standard sequence to continuously optimize and adjust the primer system, the primers are put in according to the optimized proportion shown in the following table 1, and finally, most amplification deviation is reduced to be within one time, as shown in fig. 4-5, the real distribution condition of the reaction sample is more accurate.

TABLE 1 TRB primer sets and adjustment ratios

TABLE 2 HLA-type I A, B, C amplification primer set

Because the number of different primers in the primer group in the multiplex PCR reaction system is large, in order to obtain a more ideal PCR product, the amount of the primers in the reaction system needs to be increased to 0.4mM after experiments, the reaction system configuration is carried out according to the following table 3, the mixture is shaken and uniformly mixed, and the mixture is placed in a PCR instrument after instantaneous centrifugation, and the reaction is carried out according to the following table 4.

TABLE 3 multiplex PCR reaction System

TABLE 4 multiplex PCR reaction System

4. Library construction

The amplification product was purified and recovered by magnetic column, and subjected to a second round of PCR, in which sequencing adapters and indexes of library tags were introduced, and the configuration of the PCR system is shown in Table 5 below.

TABLE 5 library add linker reaction System

Shaking and mixing evenly, placing in a PCR instrument after instantaneous centrifugation, and operating according to the conditions in the following table 6.

TABLE 6 PCR reaction conditions with addition of sequencing tags

After the reaction is finished, detecting by gel electrophoresis, and as shown in FIG. 6, performing magnetic bead purification on a plurality of band samples with obviously different lengths of 250bp-500bp in the same lane to obtain the on-machine sequencing library.

5. Sequencing on machine

The sequencing library NGS platform constructed above was programmed (here Illumina, Novaseq6000), and the sequencing type was PE 150.

6. Data analysis

The sequencing data was analyzed by immunohistochemical analysis using IMonitor software, and the analysis procedure was described in published articles (IMonitor: A Robust Pipeline for TCR and BCR repeat analysis. "Genetics201 (2015); DOI: 10.1534/genetics.115.176735).

The TRB analysis and treatment method comprises the following steps:

the sequencing data processed by the analysis flow is illustrated by taking a healthy human sample as an example, partial chart results of the data are shown after standard data analysis is carried out by using an IMonitor, as shown in fig. 7-10, fig. 7 is a schematic diagram of CDR3 length distribution, fig. 8 is a schematic diagram of CDR3 abundance distribution, fig. 9 is a schematic diagram of CDR3 frequency distribution and a schematic diagram of Top10 CDR3 frequency distribution, and fig. 10 is a schematic diagram of TRB V-J pairing three-dimensional distribution. Combining the information of Reads insert distribution, saturation curve, CDR 3V (D) J gene length distribution, V (D) J gene deletion length distribution, V (D) J gene insertion length distribution, V (D) J gene high-frequency mutation rate (base) distribution, J gene use distribution, V gene use distribution and the like. Can reflect the diversity of the immune repertoire TRB of the sample and evaluate the immunity of the sample. The sample data shown in FIGS. 7-10 is blood of healthy persons, so the sample has rich CDR3 composition and high TRB diversity in immune repertoire.

The HLA analysis processing method is as follows:

the HLA data is firstly positioned on a corresponding reference sequence (a reference sequence source IMGT/HLA database) by bwa software according to the determination result of each exon and a homologous sequence of each database is constructed, DNA in the database is screened and sequence error is corrected, finally the corrected DNA sequence and the alignment result in the database are combined with the frequency information of each exon 2,3 and 4 of A, B, C gene, and the ranking result is comprehensively analyzed, so that the HLA type information of the sample can be obtained.

The HLA-A, B, C type information obtained by genome sequencing and the like prior to this example sample was regarded as the accurate target type in this experiment, namely, the known types (A. times.24: 02:01, B. times.35: 01:01 and C. times.03: 03: 01/C. times.03: 04:01) as the control, and DNA sequence information was obtained by NGS sequencing by the TRB and HLA identical system library construction method of the present invention, and the HLA typing results are shown in Table 7 below.

TABLE 7 example sample typing results

Note: "READS" means the number of READS obtained by sequencing, "RATIO" means the RATIO among all typing classes, "RANK (Fre)" means the typing RANK (RATIO X Asian typing frequency), "EX 2" means the number of READS of exon 2, "EX 3" means the number of READS of exon 3, and "EX 4" means the number of READS of exon 4.

The result shows that bwa software is used for comparing the obtained type information of Top10 with the highest homology of the sample, and the type information ranked first or second in the list is consistent with the known type information in the sample, so that the HLA primer group in the experimental system can be judged to accurately amplify the target region of the exons 2,3 and 4 of the HLA-A, B, C gene of the sample without bias numbers and the like, HLA-I type gene sequence information is obtained through NGS sequencing, and the correct HLA type result is obtained through the processing and analysis of biological information software.

The typing results of the sample combined with TRB immune repertoire information play an important reference role in health state and disease analysis, homologous transplantation and the like.

Example 2

A kit for simultaneously detecting TCR and HLA genotyping, comprising the TCR amplification primers and HLA amplification primers of example 1.

Example 3

A system for simultaneously detecting TCR and HLA genotyping, comprising:

the detection device is used for detecting according to the method in the embodiment 1 to obtain sequencing data;

the analysis device is used for acquiring the sequencing data to analyze and process so as to obtain the TCR and HLA genotyping conditions;

and the output device is used for outputting the TCR and HLA genotyping conditions.

Example 4

And (3) verification of a method for simultaneously detecting TCR and HLA genotyping.

Samples of known HLA genotypes were selected by whole genome sequencing assay, and the assay typing was performed according to the method of example 1, and compared with the known results, which are shown in the following table.

TABLE 8 verification of comparative tests

The results show that the method for simultaneously detecting the TCR and HLA gene typing can amplify the TCR and HLA genes in the same reaction system, the same sequencing library is adopted to detect the TCR and the HLA genes, and the typing result is accurate and reliable compared with the typing result of the HLA single library construction.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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