HLA gene amplification primer based on third-generation sequencing platform and application thereof

Abstract

The application provides an HLA gene amplification primer based on a third-generation sequencing platform and an application thereof.The HLA gene amplification primer comprises a combination of any one or any multiple groups of nine groups of primers.The amplification primer designed for 11 target points of HLA not only realizes near-full-length amplification of the HLA gene, but also improves the specificity of amplification, and further improves the uniformity of HLA gene sequencing data.In addition, the amplification primer for 11 target points of the HLA gene in the application realizes one-pot detection in the same reaction system, and is convenient to operate.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid sequencing technology, and more specifically, to an HLA gene amplification primer based on a third-generation sequencing platform and its application. Background Technology

[0002] Human leukocyte antigen (HLA) is an expression product of the major histocompatibility complex in humans, located on the short arm of chromosome 6, and composed of a series of tightly linked loci. The HLA system plays a crucial role in the immune system, primarily encoding antigenic peptide presenting molecules on the cell surface. These molecules present antigenic peptides to T cell receptors (TCRs) on the surface of T cells, thereby activating the immune response.

[0003] HLA is classified into class I and class II antigens based on its distribution and function. HLA class I molecules are presenting molecules for endogenous antigens; HLA class II molecules are presenting molecules for exogenous antigens. The specificity of HLA class I antigens depends on the α heavy chain, encoded by HLA-A, B, and C loci; its β light chain is β2-microglobulin, encoded by genes on chromosome 15. HLA class II antigens are controlled by the HLA-D region (containing five subregions of nucleotide sequence), encoded by genes A and B within it, which are the α heavy chain and β light chain, respectively. Antigen polymorphism depends on the β light chain. All of these genes are polymorphic sites (multiple alleles) and co-dominant.

[0004] If we consider the MHC as a whole, its polymorphism becomes even more pronounced. Conservative estimates suggest at least 1300 different haplotypes, corresponding to approximately 17 × 10⁷ genotypes. The HLA system is one of the most polymorphic genetic systems, meaning that many different HLA gene variants (alleles) exist between different individuals. In medicine, accurate and highly precise HLA typing has important applications in organ transplant donor-recipient matching, autoimmune disease research, and forensic kinship or identity verification.

[0005] Currently, HLA testing technologies include serological typing, but this method is complex, prone to cross-reactivity, and has low accuracy and resolution. It has been gradually replaced by PCR and sequencing technologies. PCR-based HLA testing technologies mainly include Sequence-Specific Oligonucleotide Probes (SSO), Sequence-Specific Primers (SSP), Sequence-Based Typing (SBT), and Next-Generation Sequencing (NGS). SSO technology determines HLA genotype based on the hybridization signal between the probe and the PCR amplification product. However, it is cumbersome, struggles to detect new alleles, and can only differentiate known genotyping sequences, making the interpretation of nucleotide sequences somewhat subjective. SSP also fails to detect new alleles and cannot distinguish pseudogenes. SBT technology, based on first-generation sequencing, can detect new alleles but cannot distinguish between two alleles, resulting in ambiguous results. NGS, based on next-generation sequencing technology, can detect new alleles. However, the sequencing length is generally short, making it impossible to sequence the entire HLA gene. It remains limited to exons 2, 3, and 4 (the sites most studied in current technologies), and still cannot obtain sequence information of introns and UTR regions. The genotyping accuracy is generally only quartiles. Due to the extremely high polymorphism of HLA nucleotide sequences, the above technologies still have insurmountable problems, often yielding ambiguous results.

[0006] Current technologies employ third-generation sequencing (NGS) for monomeric amplification (not multiplex amplification) of class I HLA genes. This method only amplifies class I HLA genes (three HLA genes), resulting in limited HLA gene coverage. Furthermore, the single-gene locus amplification method is cumbersome and costly. Patent application CN 113817725 A utilizes the advantages of long reads from NGS and incorporates amplification regions in primer design to effectively provide complete HLA information (including exons and introns), improving the sensitivity and accuracy of genotyping. However, this method suffers from drawbacks such as excessive non-specific amplification, poor target amplification uniformity, and high library construction costs for individual samples. Therefore, developing an HLA genotyping method with strong specific amplification, high target amplification uniformity, low library construction costs, and the ability to effectively obtain complete HLA information is crucial. Summary of the Invention

[0007] The main objective of this invention is to provide an HLA gene amplification primer based on a third-generation sequencing platform and its application, in order to solve the problem of poor HLA gene amplification specificity in existing HLA gene sequencing technologies.

[0008] To achieve the above objective, according to a first aspect of the present invention, an HLA gene amplification primer is provided, the amplification primer comprising any one or more of the following nine sets of primers: wherein,

[0009] The first set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2;

[0010] The second set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 4;

[0011] The third set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 5 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 6;

[0012] The fourth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 8;

[0013] The fifth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 10;

[0014] The sixth set of primers contains an upstream primer having the nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 12;

[0015] The seventh set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 14;

[0016] The eighth set of primers contains an upstream primer having the nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 16;

[0017] The ninth set of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer having the nucleotide sequences shown in SEQ ID NO: 18 and / or SEQ ID NO: 19.

[0018] Furthermore, in the above nine primer groups, the molar ratio between the upstream and downstream primers in each primer group is (0.9~1.1):(0.9~1.1).

[0019] Further, the amplification primers are any combination of any number of the nine sets of primers; in the above composition, the molar ratio between each set of primers is: the first set of primers: the second set of primers: the third set of primers: the fourth set of primers: the fifth set of primers: the sixth set of primers: the seventh set of primers: the eighth set of primers: the ninth set of primers is (2.9~3.1): (1.9~2.1): (1.9~2.1): (8.9~9.1): (16.9~17.1): (10.9~11.1): (25.9~26.1): (3.9~4.1): (11.9~12.1).

[0020] Furthermore, the 5' end of the above amplification primers contains a tag sequence.

[0021] Furthermore, the above tag sequence is a 6-10 bp oligonucleotide sequence.

[0022] To achieve the above objectives, according to a second aspect of the present invention, a kit is provided, the kit comprising the amplification primers described above.

[0023] Furthermore, the above kit also includes any one or more of the following reagents: PCR amplification reagents, gene purification reagents, sequencing library construction reagents, or sequencing reagents; wherein the above PCR amplification reagents include: water, dNTPs, DNA polymerase, and DNA polymerase buffer.

[0024] Furthermore, the DNA polymerase mentioned above is selected from any of the following: KOD FX Neo, KOD-Multi & Epi-, KOD FX, KOD One™ PCR Master Mix, or KOD-Plus- Neo.

[0025] To achieve the above objective, according to a third aspect of the present invention, a method for HLA gene PCR amplification is provided, the method comprising: performing PCR amplification using the above-described amplification primers to obtain the above-described HLA gene amplification product.

[0026] Furthermore, the PCR amplification reaction procedure described above can be selected from any of the following:

[0027] 1) 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*4 cycles, (98℃ for 10 s, 72℃ for 12 min)*4 cycles, (98℃ for 10 s, 70℃ for 12 min)*4 cycles, (98℃ for 10 s, 68℃ for 12 min)*18 cycles, 68℃ for 7 min, store at 4℃;

[0028] 2) 94℃ for 2 min, (98℃ for 10 s, 72℃ for 12 min)*2 cycles, (98℃ for 10 s, 70℃ for 12 min)*2 cycles, (98℃ for 10 s, 68℃ for 12 min)*21 cycles, 68℃ for 7 min, store at 4℃;

[0029] 3) 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*1 cycle, (98℃ for 10 s, 73℃ for 12 min)*1 cycle, (98℃ for 10 s, 72℃ for 12 min)*1 cycle, (98℃ for 10 s, 71℃ for 12 min)*1 cycle, (98℃ for 10 s, 70℃ for 12 min)*1 cycle, (98℃ for 10 s, 69℃ for 12 min)*1 cycle, (98℃ for 10 s, 68℃ for 12 min)*21 cycles, 68℃ for 7 min, store at 4℃.

[0030] To achieve the above objectives, according to a fourth aspect of the present invention, a method for constructing an HLA gene sequencing library is provided, the method comprising: ligating an HLA gene amplification product to a sequencing adapter to obtain the above-mentioned HLA gene sequencing library;

[0031] The amplification products mentioned above are selected from the amplification products obtained by amplifying the HLA gene using the HLA gene amplification primers mentioned above or the amplification products obtained by amplifying the HLA gene using the HLA gene PCR amplification method mentioned above.

[0032] Furthermore, before ligating the amplification product to the sequencing adapter, the construction method further includes purifying the amplification product.

[0033] To achieve the above objectives, according to a fourth aspect of the present invention, an HLA gene sequencing method is provided, the HLA gene sequencing method comprising: sequencing a sequencing library; wherein the sequencing library is a sequencing library constructed using the above-described HLA gene sequencing library construction method.

[0034] Furthermore, the above sequencing employed third-generation sequencing technology.

[0035] To achieve the above objectives, according to a fifth aspect of the present invention, an HLA genotyping detection method is provided, the HLA genotyping detection method comprising: detecting and typing an HLA genotype using the above-described HLA gene sequencing method.

[0036] To achieve the above objectives, according to a sixth aspect of the present invention, the application of the above-described amplification primers or the above-described kit in the preparation of HLA genotyping detection reagents is provided.

[0037] To achieve the above objectives, according to a seventh aspect of the present invention, the application of the above-described amplification primers, kits, methods for HLA gene PCR amplification, methods for constructing HLA gene sequencing libraries, methods for HLA gene sequencing, or methods for HLA genotyping detection in HLA genotyping is provided.

[0038] Furthermore, the above HLA genotyping includes the following steps in sequence: HLA gene amplification and HLA gene sequencing library.

[0039] The present invention employs amplification primers or combinations thereof designed for 11 gene loci (including HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5), specifically containing the nucleotide sequences shown in SEQ ID NOs: 1-19. By using the aforementioned amplification primers or combinations thereof to specifically amplify and enrich HLA genes, and then performing library construction and sequencing on the amplified fragments, complete information of the HLA genes (including exons and introns) can be effectively obtained, achieving near full-length amplification of the HLA genes.

[0040] The amplification primers designed in this invention have similar annealing temperatures, which improves the enrichment specificity of HLA gene target sequences and reduces the proportion of invalid data. Furthermore, using the amplification primers in this invention's amplification program enables the simultaneous amplification of multiple genes in a single tube system, improving the homogeneity between targets and thus increasing data utilization and reducing redundancy in subsequent sequencing data. After subsequent multi-sample mixed library construction (library preparation) and gene sequencing, the data homogeneity among the samples is good. This improves the resolution of HLA gene sequencing data genotyping, achieving 6-interval resolution for HLA genotyping. Attached Figure Description

[0041] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0042] Figure 1 A schematic diagram of the amplification product peaks of the HLA-A gene locus according to Example 1 of the present invention is shown.

[0043] Figure 2 A schematic diagram of the amplification product peaks of the HLA-B gene locus according to Example 1 of the present invention is shown.

[0044] Figure 3A schematic diagram of the amplification product peaks of the HLA-C gene locus according to Example 1 of the present invention is shown.

[0045] Figure 4 A schematic diagram of the amplification product peaks of the HLA-DPA1 gene locus according to Example 1 of the present invention is shown.

[0046] Figure 5 A schematic diagram of the amplification product peaks of the HLA-DPB1 gene locus according to Example 1 of the present invention is shown.

[0047] Figure 6 A schematic diagram of the amplification product peaks of the HLA-DQA1 gene locus according to Example 1 of the present invention is shown.

[0048] Figure 7 A schematic diagram of the amplification product peaks of the HLA-DQB1 gene locus according to Example 1 of the present invention is shown.

[0049] Figure 8 A schematic diagram of the amplification product peaks of the HLA-DRB-E1 gene locus according to Example 1 of the present invention is shown.

[0050] Figure 9 A schematic diagram of the amplification product peaks of the HLA-DRB-E2~6 gene loci according to Example 1 of the present invention is shown.

[0051] Figure 10 A schematic diagram of the amplification product peaks of the HLA-A gene locus according to Comparative Example 1 of the present invention is shown.

[0052] Figure 11 A schematic diagram of the amplification product peaks of the HLA-B gene locus according to Comparative Example 1 of the present invention is shown.

[0053] Figure 12 A schematic diagram of the amplification product peaks of the HLA-C gene locus according to Comparative Example 1 of the present invention is shown.

[0054] Figure 13 A schematic diagram of the amplification product peaks of the HLA-DPA1 gene locus according to Comparative Example 1 of the present invention is shown.

[0055] Figure 14 A schematic diagram of the amplification product peaks of the HLA-DPB1 gene locus according to Comparative Example 1 of the present invention is shown.

[0056] Figure 15 A schematic diagram of the amplification product peaks of the HLA-DQA1 gene locus according to Comparative Example 1 of the present invention is shown.

[0057] Figure 16A schematic diagram of the amplification product peaks of the HLA-DQB1 gene locus according to Comparative Example 1 of the present invention is shown.

[0058] Figure 17 A schematic diagram of the amplification product peaks of the HLA-DRB-E1 gene locus according to Comparative Example 1 of the present invention is shown.

[0059] Figure 18 A schematic diagram of the amplification product peaks of the HLA-DRB-E2E6 gene locus according to Comparative Example 1 of the present invention is shown.

[0060] Figure 19 A schematic diagram showing the average sequencing depth percentage according to Embodiment 2 and Comparative Example 2 of the present invention is shown.

[0061] Figure 20 A schematic diagram of the primer amplification region for the HLA gene coding site of this invention is shown. Detailed Implementation

[0062] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0063] Terminology Explanation:

[0064] Multiplex PCR amplification: A PCR technique that simultaneously amplifies multiple target sequences in the same reaction system. It can be used to simultaneously detect multiple genes or DNA fragments, saving time and cost and improving experimental efficiency. Multiplex PCR amplification typically requires designing multiple primers corresponding to different target sequences and optimizing PCR reaction conditions to ensure that each target sequence is effectively amplified. This technique is widely used in molecular biology research, disease diagnosis, and genetic analysis.

[0065] MHC stands for major histocompatibility complex, which is a highly polymorphic genome consisting of a group of chromosomes that encode proteins related to intercellular recognition and antigen presentation. In humans, it is called the HLA complex.

[0066] Alleles: These are different gene forms at the same locus that perform the same function but express it in different ways. In an individual, each locus can have two alleles, one from the mother and one from the father.

[0067] HLA-DRB1 / 3 / 4 / 5: These belong to class II HLA genes. HLA-DRB1 encodes the β chain of the HLA-DR molecule. This β chain forms a heterodimer with the α chain encoded by HLA-DRA; both are anchored in the membrane and participate in antigen presentation. HLA-DRB3 / 4 / 5 have high homology with HLA-DRB1 and also form heterodimers with HLA-DRA1 for cell surface display of polypeptide antigens. However, the protein expression levels of HLA-DRB3 / 4 / 5 are low; for example, the expression level of HLA-DRB5 is only 20% of that of HLA-DRB1. HLA-DRB3 / 4 / 5 are located adjacent to HLA-DRB1, which is carried by all individuals and is located in the core region of the HLA-DR locus (p21.3 on the short arm of chromosome 6). Individuals with HLA-DRB3 / 4 / 5 may carry 0, 1, or 2 of the genes (i.e., each haplotype carries at most one DRB3 / 4 / 5 gene), but they usually do not carry multiple genes from DRB3, DRB4, and DRB5 simultaneously. Moreover, the specific locus is strongly correlated with the HLA-DRB1 genotype, and there is extremely strong linkage disequilibrium between HLA-DRB1 and HLA-DRB3 / 4 / 5.

[0068] A gene locus is a specific location on a chromosome that corresponds to a particular gene. Each gene locus can contain different alleles, and these alleles determine an individual's genetic characteristics. By studying the combination and distribution of alleles at gene loci, we can understand an individual's genetic information and phenotypic traits. Gene loci play a crucial role in genetic research, used to explore issues such as genetic variation, genome associations, and evolution.

[0069] Third-generation sequencing technology refers to single-molecule real-time sequencing technology. Compared with the first two generations of sequencing technologies, its biggest feature is that the sequencing process of single-molecule sequencing does not require PCR amplification, realizing the sequencing of each DNA molecule individually. Currently, the main third-generation sequencing technologies can be divided into nanopore electro-signal sequencing and single-molecule fluorescence signal sequencing according to different sequencing principles. Specifically, the main third-generation sequencing technologies can be divided into: Oxford Nanopore Technologies (ONT)'s single-molecule nanopore DNA sequencing technology and Pacific Biosciences (PacBio)'s single-molecule real-time (SMRT) technology.

[0070] As mentioned in the background section, existing HLA gene sequencing technologies suffer from poor amplification specificity and sequencing uniformity. In this invention, the inventors designed specific primers with similar annealing temperatures targeting 11 HLA target sites and employed gradient annealing to improve the amplification specificity and sequencing uniformity of HLA gene detection, thus proposing the protection scheme of this invention.

[0071] In a first typical embodiment of the present invention, an HLA gene amplification primer is provided, the amplification primer comprising any one or any combination of any one or more of the following nine sets of primers: wherein,

[0072] The first set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2;

[0073] The second set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 4;

[0074] The third set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 5 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 6;

[0075] The fourth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 8;

[0076] The fifth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 10;

[0077] The sixth set of primers contains an upstream primer having the nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 12;

[0078] The seventh set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 14;

[0079] The eighth set of primers contains an upstream primer having the nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 16;

[0080] The ninth set of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer having the nucleotide sequences shown in SEQ ID NO: 18 and / or SEQ ID NO: 19.

[0081] The HLA gene includes multiple coding sites: HLA-A (first primer group), HLA-B (second primer group), HLA-C (third primer group), HLA-DPA1 (fourth primer group), HLA-DPB1 (fifth primer group), HLA-DQA1 (sixth primer group), HLA-DQB1 (seventh primer group), HLA-DRB-E1 (eighth primer group), and HLA-DRB-E2~6 (ninth primer group). See details... Figure 20 .

[0082] It should be noted that the HLA-DRB gene family includes four genotypes: HLA-DRB1, HLA-DRB3, HLA-4, and HLA-5. Because these four genotypes share significant sequence similarity and all contain six exons, the same primer set is used for amplification of these four genotypes. Specifically, primer set eight targets the first exon region, and primer set nine targets exons 2 through 6.

[0083] Furthermore, the compositions in the above-mentioned "any number of compositions" include mixed phases as well as single phases sold or used in sets.

[0084] In PCR amplification systems, primer annealing design is one of the key factors affecting amplification specificity. Designing primers with an annealing temperature higher than the actual reaction temperature will lead to non-specific binding to the template, resulting in non-specific amplification. Designing primers with an annealing temperature lower than the actual reaction temperature will result in incomplete annealing and low amplification efficiency. In multiplex amplification systems, multiple sets of primers are amplified under the same conditions and using the same annealing temperature. Differences in annealing temperatures between different primers can exacerbate non-specific amplification and differences in amplification efficiency, making the design of multiplex amplification programs complex.

[0085] In multiplex PCR amplification, by adjusting primer sequence length and GC content, and designing reasonable amplification primer sequences to ensure similar annealing temperatures, combined with a gradient-fall PCR amplification program, the specificity of the primer system can be enhanced while maintaining amplification efficiency. The nine primer sets mentioned above were designed with similar annealing temperatures (60℃~65℃), and using the same PCR amplification conditions resulted in high primer specificity. Using these amplification primers to amplify the HLA gene helps to ensure amplification efficiency while improving the specificity of the amplification system.

[0086] The above nine primers are mixed and then used for multiplex PCR amplification in a single system (one tube), which can cover all coding sites of the HLA gene. Through subsequent library construction and sequencing, the target region carrying all information can be obtained, and HLA genotyping can be accurately detected.

[0087] Multiple primer sets are used for amplification within a single tube. Competition for substrate amplification arises between primers, leading to variations in amplification folds and poor target coverage uniformity. Some targets may experience insufficient amplification, requiring further increases in sequencing data to improve coverage depth, thus raising sequencing costs. Conversely, some targets may suffer from over-amplification, potentially causing data redundancy. Therefore, differences in amplification uniformity within the same system can affect sequencing coverage depth, further impacting the uniformity of sequencing data. By adjusting the amount of HLA primers used in multiplex amplification, combined with the primers designed in this invention and the corresponding PCR amplification system, amplification uniformity and data utilization can be improved, thereby enhancing the coverage uniformity and data utilization of sequencing data.

[0088] The molar ratio of the upstream primer and its corresponding downstream primer within a primer set may affect the amplification efficiency of the target gene. In a preferred embodiment of the present invention, the molar ratio of the upstream primer to the downstream primer within each of the nine primer sets is (0.9~1.1):(0.9~1.1). In a more preferred embodiment of the present invention, the molar ratio of the upstream primer to the downstream primer within each of the nine primer sets is 1:1. PCR amplification using upstream and downstream primers with the above molar ratios offers the beneficial effects of high amplification efficiency and good uniformity.

[0089] It should be noted that the ninth primer group provides two downstream primers, with sequences SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In a preferred embodiment of the present invention, the ninth primer group comprises an upstream primer with the nucleotide sequence SEQ ID NO: 17 and a downstream primer with the nucleotide sequence SEQ ID NO: 18 and / or a downstream primer with the nucleotide sequence SEQ ID NO: 19. That is, the two downstream primers can be used simultaneously or either one can be used at will.

[0090] In a more preferred embodiment of the present invention, the ninth set of primers comprises an upstream primer with the nucleotide sequence SEQ ID NO: 17, a downstream primer with the nucleotide sequence SEQ ID NO: 18, and a downstream primer with the nucleotide sequence SEQ ID NO: 19. In a further preferred embodiment of the present invention, the molar ratio of the downstream primer with the nucleotide sequence SEQ ID NO: 18 to the downstream primer with the nucleotide sequence SEQ ID NO: 19 is 1:1. Simultaneously using the above two types of downstream primers can cover different variation types in polymorphic regions, adapting to complex gene structures. In the case of highly polymorphic or complex gene structures, targeting different specific sites, it has the beneficial effects of reducing non-specific amplification and improving the accuracy, reliability, and amplification efficiency of PCR results.

[0091] In a preferred embodiment of the present invention, the amplification primers are a combination of any number of sets of nine primers; the molar ratio between each set of primers in the composition is: first set of primers: second set of primers: third set of primers: fourth set of primers: fifth set of primers: sixth set of primers: seventh set of primers: eighth set of primers: the ninth set of primers is (2.9~3.1): (1.9~2.1): (1.9~2.1): (8.9~9.1): (16.9~17.1): (10.9~11.1): (25.9~26.1): (3.9~4.1): (11.9~12.1).

[0092] Using the above nine primer sets in molar ratio for multiplex PCR amplification can effectively improve the uniformity of target sequence coverage in the amplification products, thereby helping to improve the effective utilization rate of subsequent sequencing data and reduce sequencing costs.

[0093] In a more preferred embodiment of the present invention, the molar ratio of the working concentrations of each primer set is: Primer set 1: Primer set 2: Primer set 3: Primer set 4: Primer set 5: Primer set 6: Primer set 7: Primer set 8: Primer set 9 = 3:2:2:9:17:11:26:4:12. Using multiple sets of amplification primers with the above molar ratio for multiplex PCR helps to improve the uniformity of amplification.

[0094] In order to improve sequencing efficiency when performing HLA gene sequencing on multiple samples simultaneously, in a preferred embodiment of the present invention, the 5' end of the amplification primers contains a tag sequence. In a more preferred embodiment of the present invention, the tag sequence is 6-10 bases. In a further preferred embodiment of the present invention, the tag sequence is 6-8 bases.

[0095] In a preferred embodiment of the present invention, the tag sequences used for multiple samples that are co-constructed for library preparation or co-sequencing contain the same GC content. In a more preferred embodiment of the present invention, there are no inverse complementary sequences between the aforementioned tag sequences and the primer sequences within the nine groups. The aforementioned tag sequences with the same GC content can avoid the influence of sequencing bias and batch effects on amplification efficiency and the uniformity of sequencing data, ensuring the balance of sequencing signals and thus improving the accuracy of genotyping results.

[0096] For simultaneous sequencing of multiple samples, amplification products with different tag sequences can be directly mixed during library construction. Different samples carry different tag sequences, allowing sequencing data to be split according to the sample after sequencing using different tag information. Using amplification primers with tag sequences can improve the throughput of mixed library construction sequencing, offering advantages such as convenience, efficiency, and cost reduction. The tag sequence can be any combination of sequences that does not affect amplification and / or library construction operations.

[0097] In a second typical embodiment of the present invention, a kit is provided, which includes the amplification primers described above. In a preferred embodiment of the present invention, the kit further includes any one or more of the following reagents: PCR amplification reagents, gene purification reagents, sequencing library construction reagents, and sequencing reagents; wherein the PCR amplification reagents include: water, dNTPs, DNA polymerase, and DNA polymerase buffer. Using the above kit for HLA genotyping has the advantage of being simple to operate. It should be noted that the gene purification reagents, sequencing library construction reagents, and sequencing reagents described above are all reagents commonly used in the art.

[0098] In a preferred embodiment of the present invention, the DNA polymerase is selected from any one of the following: KOD FX Neo, KOD-Multi & Epi-, KOD FX, KOD One™ PCR Master Mix, or KOD-Plus- Neo. Using the above-mentioned high-fidelity DNA polymerase can effectively improve the accuracy and consistency of the amplification products, thus contributing to improved accuracy in HLA genotyping.

[0099] In a third typical embodiment of the present invention, a method for HLA gene PCR amplification is provided. This method includes: performing PCR amplification using the aforementioned amplification primers to obtain HLA gene amplification products. Using the above method to amplify the HLA gene can improve the amplification heterogeneity and efficiency, thereby making the amplification of the HLA gene more accurate.

[0100] In a preferred embodiment of the present invention, the PCR amplification reaction procedure is selected from any of the following:

[0101] 1) 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*4 cycles, (98℃ for 10 s, 72℃ for 12 min)*4 cycles, (98℃ for 10 s, 70℃ for 12 min)*4 cycles, (98℃ for 10 s, 68℃ for 12 min)*18 cycles, 68℃ for 7 min, store at 4℃;

[0102] 2) 94℃ for 2 min, (98℃ for 10 s, 72℃ for 12 min)*2 cycles, (98℃ for 10 s, 70℃ for 12 min)*2 cycles, (98℃ for 10 s, 68℃ for 12 min)*21 cycles, 68℃ for 7 min, store at 4℃;

[0103] 3) One cycle of 94℃ for 2 min, followed by (98℃ for 10 s, 74℃ for 12 min), then another cycle of (98℃ for 10 s, 73℃ for 12 min), then another cycle of (98℃ for 10 s, 72℃ for 12 min), then another cycle of (98℃ for 10 s, 71℃ for 12 min), then another cycle of (98℃ for 10 s, 70℃ for 12 min), then another cycle of (98℃ for 10 s, 69℃ for 12 min), then another cycle of (98℃ for 10 s, 68℃ for 12 min), followed by 21 cycles of (98℃ for 10 s, 68℃ for 12 min), then a final 7 min at 68℃, followed by storage at 4℃. This PCR amplification program helps suppress non-specific amplification, enrich target regions, and improve amplification specificity.

[0104] In a more preferred embodiment of the present invention, the PCR amplification reaction program is selected from the above 1) 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*4 cycles, (98℃ for 10 s, 72℃ for 12 min)*4 cycles, (98℃ for 10 s, 70℃ for 12 min)*4 cycles, (98℃ for 10 s, 68℃ for 12 min)*18 cycles, 68℃ for 7 min, and stored at 4℃.

[0105] In a fourth typical embodiment of the present invention, a method for constructing an HLA gene sequencing library is provided. This method includes: ligating HLA gene amplification products to sequencing adapters to obtain an HLA gene sequencing library; the amplification products are selected from those obtained by amplifying the HLA gene using the aforementioned HLA gene amplification primers or those obtained by amplifying the HLA gene using the aforementioned HLA gene PCR amplification method. Using the above-described method for constructing an HLA gene sequencing library helps improve the quality and depth of sequencing data, providing a data foundation for high-resolution HLA genotyping.

[0106] In a preferred embodiment of the present invention, before ligating the amplification product and the sequencing adapter, the construction method further includes purifying the amplification product. The purpose of purification is to remove reagents and non-target products from the PCR reaction system. In a more preferred embodiment of the present invention, the purification method is selected from any one or more of the following: magnetic bead method or silica gel column method.

[0107] In a fifth typical embodiment of the present invention, an HLA gene sequencing method is provided. This method includes sequencing a sequencing library; the sequencing library is a sequencing library constructed using the above-described HLA gene sequencing library construction method. In a preferred embodiment of the present invention, sequencing employs third-generation sequencing technology. The sequencing data obtained using the above-described sequencing method exhibits high capture specificity and strong data uniformity, which helps improve the accuracy of HLA genotyping, increase detection efficiency, and reduce detection costs. Third-generation sequencing technology, with its long read lengths and high throughput, can provide more comprehensive HLA gene information, thereby helping to improve the accuracy of HLA genotyping.

[0108] In a sixth typical embodiment of the present invention, an HLA genotyping detection method is provided. The HLA genotyping detection method includes: detecting and typing the HLA genotype using the aforementioned HLA gene sequencing method. This method provides high accuracy for HLA genotyping and can improve the precision of HLA genotyping.

[0109] In a seventh typical embodiment of the present invention, the application of the above-mentioned amplification primers or the above-mentioned kit in the preparation of HLA genotyping detection reagents is provided.

[0110] In the eighth typical embodiment of the present invention, the application of the above-mentioned amplification primers, kits, HLA gene PCR amplification methods, HLA gene sequencing library construction methods, HLA gene sequencing methods, or HLA genotyping detection methods in HLA genotyping is provided.

[0111] In a preferred embodiment of the present invention, HLA genotyping includes the following steps in sequence: HLA gene amplification, HLA gene sequencing library construction, HLA gene sequencing, and HLA genotyping determination.

[0112] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0113] Example 1: Specificity of single primers and amplification systems

[0114] I. PCR Amplification

[0115] The primer sequences designed for different HLA gene targets are shown in Table 1 below:

[0116] Table 1

[0117]

[0118] In the table, F represents the forward primer, also known as the upstream primer; R represents the reverse primer, also known as the downstream primer. Furthermore, two reverse primers were designed for HLA-DRB-E2~6 to cover different variant types in the polymorphic region and adapt to complex gene structures. In cases of high polymorphism or complex gene structures, targeting different specific sites reduces the possibility of non-specific amplification, improving the accuracy, reliability, and amplification efficiency of PCR results.

[0119] The PCR amplification system is shown in Table 2 below:

[0120] Table 2

[0121]

[0122] Note: NA12878 gDNA (human genomic DNA standard). It should be noted that all human genomic DNA standards used in this invention were purchased from the Coriell Institute. The HLA-Prim in the table includes upstream and downstream primers, wherein the molar ratio of upstream and downstream primers targeting the same gene target is 1:1. Specifically, the molar ratio of the upstream primer and the two downstream primers targeting HLA-DRB-E2~6 is F:R1:R2 = 2:1:1.

[0123] PCR program 1 is as follows: 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*4 cycles, (98℃ for 10 s, 72℃ for 12 min)*4 cycles, (98℃ for 10 s, 70℃ for 12 min)*4 cycles, (98℃ for 10 s, 68℃ for 12 min)*18 cycles, 68℃ for 7 min, store at 4℃.

[0124] II. Purification

[0125] 1. Add 1.0 volume of magnetic beads (VAHTS DNA Clean Beads) to the reaction system, mix well, and let stand at room temperature for 8 minutes;

[0126] 2. Place on a magnetic rack and let stand for 5 minutes until the magnetic beads are completely attracted, then remove the supernatant;

[0127] 3. Add 200 µL of freshly prepared 80% ethanol, let stand for 30 seconds, remove the supernatant, and repeat the washing once;

[0128] 4. As for the handheld centrifuge, it is used to remove residual alcohol;

[0129] 5. Add 20 µL of EB buffer to resuspend, and let stand at room temperature for 5 minutes;

[0130] 6. Place on a magnetic rack, let stand for 3 minutes, and aspirate the supernatant to obtain the amplification product.

[0131] III. Testing

[0132] The fragment distribution of the amplified products was checked using an Agilent 2100 bioanalyzer.

[0133] Experimental results:

[0134] Figure 1-9 The peak diagrams are for the amplified products of HLA-A (F / R), HLA-B (F / R), HLA-C (F / R), HLA-DPA1 (F / R), HLA-DPB1 (F / R), HLA-DQA1 (F / R), HLA-DQB1 (F / R), HLA-DRB-E1 (F / R), and HLA-DRB-E2E6 (F / R1+R2) in Example 1 (SEQ ID NOs: 1~19).

[0135] Comparative Example 1

[0136] This comparative example uses the gene amplification primers from patent application CN 113817725 A for the experiment.

[0137] I. Amplification

[0138] The primer sequences are shown in Table 3 below:

[0139] Table 3

[0140]

[0141] The PCR reaction system is the same as Table 2 in Example 1;

[0142] PCR program 4 is as follows: 94℃ for 2 min, (98℃ for 10 s, 68℃ for 12 min <starting from the 11th cycle, each cycle increases by 30 s>) * 30 cycles, 68℃ for 10 min, store at 4℃.

[0143] II. Purification and Detection

[0144] The purification steps and methods for detecting the distribution of amplified product fragments are the same as in Example 1.

[0145] Experimental results:

[0146] Figure 10-18The peak diagrams are for the amplification products of the primers (SEQ ID NOs: 20~37) in the comparative examples: HLA-A(F / R), HLA-B(F / R), HLA-C(F / R), HLA-DPA1(F / R), HLA-DPB1(F / R), HLA-DQA1(F / R), HLA-DQB1(F / R), HLA-DRB-E1(F / R) and HLA-DRB-E2E6(F / R1+R2).

[0147] Analysis of experimental results:

[0148] Experimental results show that, compared with the peak diagram of the amplification product of the corresponding target primer in Comparative Example 1 ( Figure 10-18 In Example 1, the amplification product peak diagrams of the primers designed for each target site in this invention are shown. Figure 1-9 The non-specific amplification product peaks other than the main peak have lower signals, indicating that the amplification specificity of each target primer designed in Example 1 of this invention has been significantly improved.

[0149] Example 2: Multiplex primer amplification system and PCR procedure

[0150] I. Amplification

[0151] Using the primer sequences designed for different targets in Table 1, diluted with low TE buffer, and then mixed according to the proportions shown in Table 4 below, this mixture is named the primer mix. The molar ratio of upstream and downstream primers targeting the same gene target is 1:1. Specifically, the molar ratio of the upstream primer and the two downstream primers targeting HLA-DRB-E2E6 is F:R1:R2 = 2:1:1.

[0152] Table 4

[0153]

[0154] Prepare the PCR amplification system according to Table 5 below:

[0155] Table 5

[0156]

[0157] The PCR reaction procedure is as follows:

[0158] PCR program 1 (same as Example 1): 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*4 cycles, (98℃ for 10 s, 72℃ for 12 min)*4 cycles, (98℃ for 10 s, 70℃ for 12 min)*4 cycles, (98℃ for 10 s, 68℃ for 12 min)*18 cycles, 68℃ for 7 min, store at 4℃.

[0159] PCR program 2: 94℃ for 2 min, (98℃ for 10 s, 72℃ for 12 min)*2 cycles, (98℃ for 10 s, 70℃ for 12 min)*2 cycles, (98℃ for 10 s, 68℃ for 12 min)*21 cycles, 68℃ for 7 min, store at 4℃.

[0160] PCR program 3: 94℃ 2min, (98℃ 10s, 74℃ 12min)*1 cycle, (98℃ 10s, 73℃ 12min)*1 cycle, (98℃ 10s, 72℃ 12min)*1 cycle, (98℃ 10s, 71℃ 12min)*1 cycle, (98℃ 10s, 70℃ 12min)*1 cycle, (98℃ 10s, 69℃ 12min)*1 cycle, (98℃ 10s, 68℃ 12min)*21 cycles, 68℃ 7min, store at 4℃.

[0161] PCR program 4 (same as control example 1): 94℃ 2min, (98℃ 10s, 68℃ 12min <starting from the 11th cycle, each cycle increases by 30s>) * 30 cycles, 68℃ 10min, store at 4℃.

[0162] II. Purification

[0163] The purification steps are the same as in Example 1.

[0164] III. Library Construction and Sequencing

[0165] Library construction was performed using the PacBio SMRTbell prep kit 3.0 library construction protocol. Fragment distribution was checked using the Agilent Femto P µLse system. Sequencing was performed according to the PacBio Revio recommended protocol and reagents.

[0166] Comparative Example 2: Multiple Primer Amplification

[0167] This comparative example uses the gene amplification primers from patent application CN 113817725 A. The designed primer sequences are the same as those in Table 3 of Comparative Example 1.

[0168] Mix the primers in Table 3 according to the scheme in Table 6 below (named "total primer"). The molar ratio of upstream and downstream primers targeting the same gene target is 1:1. The molar ratio of the upstream primer and the two downstream primers targeting HLA-DRB-E2~6 is F:R1:R2 = 2:1:1.

[0169] Table 6

[0170]

[0171] The PCR reaction system is shown in Table 7 below:

[0172] Table 7

[0173]

[0174] The PCR reaction procedure was the same as that of control group 1.

[0175] II. Purification

[0176] The purification steps are the same as in Example 1.

[0177] III. Library Construction and Sequencing

[0178] The library construction and sequencing steps are the same as in Example 2.

[0179] Experimental results: See Tables 8 and 9 below. Figure 19 .

[0180] Table 8

[0181]

[0182] Table 9:

[0183]

[0184] It should be noted that capture specificity refers to the proportion of valid data that matches the target region out of the total data volume.

[0185] Analysis of experimental results:

[0186] 1) From the perspective of capture specificity

[0187] The results of Example 2 are shown in Tables 8 and 9. Figure 19 As shown, all 11 sites were detected normally within the same system. Compared to Comparative Example 2, the groups using the amplification primers of this invention exhibited higher capture specificity, for example, Example 2-PCR program 1, Example 2-PCR program 2, Example 2-PCR program 3, and Example 2-PCR program 4. This demonstrates that the primers designed in this invention have superior capture specificity for multiplex amplification within the same system.

[0188] In addition, compared with PCR programs without gradient annealing (Example 2-PCR program 4 and Comparative Example 2), PCR programs with gradient annealing steps from high to low have higher capture specificity for amplification, indicating that gradient annealing has a significant promoting effect on amplification specificity, for example, Example 2-PCR program 1, Example 2-PCR program 2 and Example 2-PCR program 3.

[0189] In summary, the comparison example 2 of the PCR program 1-4 designed in this invention has improved the target capture specificity, with the specificity improved by at least 4 percentage points and at most 8 percentage points.

[0190] 2) From the perspective of uniformity of sequencing depth

[0191] Compared to Comparative Example 2, the CV values ​​(coefficient of variation) for sequencing depths of each target site in Example 2-PCR programs 1-4 of the present invention are lower, indicating that the output uniformity of the present invention is better. Among them, Example 2-PCR program 1 has the lowest CV value and the best uniformity.

[0192] For genotyping identification, each target needs to meet a certain coverage depth. That is, the target with the lowest average depth percentage determines the lower limit of data utilization. Taking the HLA-DRB-E2~E6 exon data with the lowest average sequencing depth as an example: In Example 2-PCR program 1, with the lowest average depth of HLA-DRB-E2~E6 at 5.1%, its sequencing depth is required to be at least 100×, and the total data depth to be measured is 1960× (the calculation formula is: 100× / 5.1%=1960×).

[0193] Similarly, calculating the data depth required to satisfy the target site at a depth of 100× for HLA-DRB-E2E6, which has the lowest average depth in Comparative Example 2 (0.2%), the required data depth is 50,000× (calculated as: 100× / 0.2%=50,000×). Therefore, the improved PCR program 1 in Example 2 of this invention requires only 3.9% of the data volume of Comparative Example 2 (calculated as: 1960× / 50000×=3.9%), significantly reducing sequencing costs.

[0194] Example 3: Multiplex amplification system with barcode primers and genotyping detection

[0195] The primer sequences designed for different targets are shown in Table 10 below (where the underlined NNNNNNNN represents the barcode sequence, and the rest are primer sequences):

[0196] Table 10

[0197]

[0198] The barcode sequence used in this experiment is:

[0199] Table 11

[0200]

[0201] The primers were diluted with low TE buffer and then mixed according to the proportions in Example 2, and named primermix;

[0202] The PCR amplification system was configured according to Example 2; the PCR reaction procedure was the same as in Example 1.

[0203] II. Purification

[0204] The purification steps are the same as in Example 1.

[0205] III. Library Construction and Sequencing

[0206] The six PCR products were mixed in equal quantities, and libraries were constructed using the PacBio SMRTbell prep kit 3.0 library construction protocol. Fragment distribution of the libraries was checked using the Agilent Femto Pulse system. Sequencing was performed according to the PacBio Revio recommended protocol.

[0207] Fourth, compare the sequencing data with the data recorded in the IMGT HLA database to analyze and determine the specific genotyping results.

[0208] Experimental results:

[0209] Six samples were amplified and mixed into libraries using six different barcode primers. The amount of split data is shown in the table below. The proportion of the output data was basically the same, close to the theoretical proportion of 16%, indicating that the splitting of the mixed library after amplification using barcode primers was normal.

[0210] Table 12

[0211]

[0212] As shown in Table 12, the results of library construction and sequencing data splitting for the six samples obtained using the scheme designed in this invention demonstrate that this invention has good library construction and sequencing data yield and uniformity. The sequencing data was further used to genotype the samples, and the genotyping results are shown in Tables 13-1 and 13-2.

[0213] Table 13-1

[0214]

[0215] Table 13-2

[0216]

[0217] (For reference typing, see: Bettinotti PM, Ferriola D, Duke LJ, et al. Characterization of 108 Genomic DNA Reference Materials for 11 HumanLeukocyte Antigen (HLA) Loci: A GeT-RM Collaborative Project [J]. The Journal of Molecular Diagnostics, 2018, 20 (5): 703-715.)

[0218] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0219] This invention designs primers targeting 11 HLA targets to achieve near full-length amplification of the HLA gene. The amplified products are sequenced using a third-generation sequencing platform to obtain the full-length sequence of the HLA gene, improving the resolution of HLA genotyping. It can achieve 6 quartiles with 100% genotyping accuracy. Furthermore, the amplification primers for all 11 targets are used in the same reaction system, enabling detection in a single tube.

[0220] In terms of primer design, this invention adjusts the sequence length of amplification primers to ensure they are uniformly annealed at similar temperatures, which helps improve amplification specificity. Furthermore, the PCR amplification program of this invention uses gradient annealing, amplifying from high to low annealing temperatures, which effectively suppresses non-specific amplification, enriches the amplification of target regions, and improves amplification specificity.

[0221] This invention improves the amplification uniformity of primers and thus increases data utilization by adjusting the ratio of primer usage.

[0222] By adding barcodes to the primer ends, different samples are amplified using different barcodes. After amplification of multiple samples, it is possible to construct a mixed third-generation library from multiple samples, thereby amortizing the cost of third-generation library construction, reducing the overall cost, and increasing the throughput. Simultaneously, the system design of this scheme ensures the output of mixed library construction data and the uniformity of data splitting across different samples.

[0223] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An HLA gene amplification primer, characterized in that, The amplification primers comprise a composition of the following nine primer groups: wherein... The first set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2; The second set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 4; The third set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 5 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 6; The fourth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 8; The fifth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 10; The sixth set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 12; The seventh set of primers includes an upstream primer having the nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 14; The eighth set of primers contains an upstream primer having the nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 16; The ninth set of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 18 and / or SEQ ID NO:

19.

2. The amplification primer according to claim 1, characterized in that, In the nine primer groups, the molar ratio between the upstream and downstream primers in each primer group is (0.9~1.1):(0.9~1.1).

3. The amplification primer according to claim 2, characterized in that, The amplification primers are any combination of multiple sets of the nine primer sets; the molar ratio between the primer sets in the composition is: The first set of primers: the second set of primers: the third set of primers: the fourth set of primers: the fifth set of primers: the sixth set of primers: the seventh set of primers: the eighth set of primers: the ninth set of primers is (2.9~3.1): (1.9~2.1): (1.9~2.1): (8.9~9.1): (16.9~17.1): (10.9~11.1): (25.9~26.1): (3.9~4.1): (11.9~12.1).

4. The amplification primer according to claim 1, characterized in that, The 5' end of the amplification primer contains a tag sequence.

5. The amplification primer according to claim 4, characterized in that, The tag sequence is a 6-10 bp oligonucleotide sequence.

6. A reagent kit, characterized in that, The kit includes the amplification primers as described in any one of claims 1-5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes any one or more of the following reagents: PCR amplification reagents, gene purification reagents, sequencing library construction reagents, or sequencing reagents; wherein the PCR amplification reagents include: water, dNTPs, DNA polymerase, and DNA polymerase buffer.

8. The reagent kit according to claim 7, characterized in that, The DNA polymerase is selected from any one of the following: KODFX Neo, KOD-Multi & Epi-, KOD FX, KOD One™ PCR Master Mix, or KOD-Plus- Neo.

9. A method for HLA gene PCR amplification, characterized in that, The method includes: performing PCR amplification using the amplification primers described in any one of claims 1-5 to obtain the HLA gene amplification product.

10. The method according to claim 9, characterized in that, The PCR amplification reaction procedure is selected from any of the following: 1) 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*4 cycles, (98℃ for 10 s, 72℃ for 12 min)*4 cycles, (98℃ for 10 s, 70℃ for 12 min)*4 cycles, (98℃ for 10 s, 68℃ for 12 min)*18 cycles, 68℃ for 7 min, store at 4℃; 2) 94℃ for 2 min, (98℃ for 10 s, 72℃ for 12 min)*2 cycles, (98℃ for 10 s, 70℃ for 12 min)*2 cycles, (98℃ for 10 s, 68℃ for 12 min)*21 cycles, 68℃ for 7 min, store at 4℃; 3) 94℃ for 2 min, (98℃ for 10 s, 74℃ for 12 min)*1 cycle, (98℃ for 10 s, 73℃ for 12 min)*1 cycle, (98℃ for 10 s, 72℃ for 12 min)*1 cycle, (98℃ for 10 s, 71℃ for 12 min)*1 cycle, (98℃ for 10 s, 70℃ for 12 min)*1 cycle, (98℃ for 10 s, 69℃ for 12 min)*1 cycle, (98℃ for 10 s, 68℃ for 12 min)*21 cycles, 68℃ for 7 min, store at 4℃.

11. A method for constructing an HLA gene sequencing library, characterized in that, The construction method includes: ligating the amplification product of the HLA gene with a sequencing adapter to obtain the HLA gene sequencing library; The amplification product is selected from the amplification product obtained by amplifying the HLA gene using the HLA gene amplification primers according to any one of claims 1-5, or the amplification product obtained by amplifying the HLA gene using the HLA gene PCR amplification method according to claim 9 or 10.

12. The construction method according to claim 11, characterized in that, Before ligating the amplification product to the sequencing adapter, the construction method further includes purifying the amplification product.

13. An HLA gene sequencing method, characterized in that, The HLA gene sequencing method includes: sequencing a sequencing library; the sequencing library is a sequencing library constructed using the HLA gene sequencing library construction method of claim 11 or 12.

14. The sequencing method according to claim 13, characterized in that, The sequencing used third-generation sequencing technology.

15. A method for HLA genotyping detection, characterized in that, The HLA genotyping detection method includes: detecting and typing the HLA genotype using the HLA gene sequencing method described in claim 13 or 14.

16. The use of any one of the amplification primers of claims 1-5 or any one of the kits of claims 6-8 in the preparation of HLA genotyping reagents.

17. The application of the amplification primers of any one of claims 1-5, the kit of any one of claims 6-8, the method for HLA gene PCR amplification of any one of claims 9-10, the method for constructing an HLA gene sequencing library as described in claim 11 or 12, the HLA gene sequencing method as described in claim 13 or 14, or the HLA genotyping detection method as described in claim 15 in HLA genotyping.

18. The application according to claim 17, characterized in that, The HLA genotyping process includes the following steps in sequence: HLA gene amplification, HLA gene sequencing library construction, HLA gene sequencing, and HLA genotyping determination.

Images