HLA gene amplification primers, kit and sequencing library construction method

Abstract

The present application is applicable to the field of gene detection technology, and provides HLA gene amplification primers, a kit, and a sequencing library construction method. The 16 sets of HLA gene multiple amplification primer sets provided in the present application can be individually PCR amplified to obtain amplified genes, and can be combined for single-tube multiple PCR amplification, thereby simplifying the experimental process and improving the sensitivity and accuracy of typing. The preparation of the library using the primer set amplification product of the present application can be applied to the detection of six genome alleles based on the sequencing technology of the second-generation sequencing platform, and has the advantages of a short detection cycle and can meet the accuracy of six-point typing, solving the problems of low accuracy and long detection cycle of existing detection technologies.

Description

Technical Field

[0001] The present application belongs to the field of gene detection technology, and in particular to an HLA gene multiple amplification primer set, a kit, and a sequencing library construction method. Background Art

[0002] Human Leukocyte Antigen (HLA) is a class of genes encoding the Major Histocompatibility Complex (MHC) on chromosome 6. HLA has become an important genetic marker for humans due to its high polymorphism and plays an important role in immune response and regulation.

[0003] Hematopoietic stem cell transplantation can be used to treat a variety of blood diseases, including hematological malignancies such as acute leukemia, chronic myeloid leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, etc.; as well as some non-malignant hematological tumors such as severe aplastic anemia and thalassemia. About 60,000 patients worldwide receive hematopoietic stem cell transplantation each year, and the fate of these patients depends on how clinicians can quickly find a well-matched donor for them through HLA testing. HLA-I type (HLA-A, B, C) and HLA-II type (HLA-DPA1, DPB1, DQA1, DQB1, DRA, DRB1, DRB3 / 4 / 5) both play an important role in transplant matching.

[0004] With the development of sequencing technology, DNA sequence-based HLA typing technology has replaced traditional serological and cytological typing methods, including specific primer method (SSP), sequence-specific oligonucleotide probe method (SSO), first-generation sequencing method (SBT) and nearly second-generation sequencing method (NGS) genotyping. SBT typing is the standard for HLA typing recommended by WHO and has become the "gold standard" for high-resolution HLA testing. In 2017, the HLA (A, B, C, DQB1, DRB1) typing kit (PCR-SBT method) produced by TBG Biotech Co., Ltd. obtained CFDA certification. This product does not contain DPB1 and other Class II locus typing reagents, and cannot type other classic HLA genes. However, studies have confirmed that DPB1 mismatch greatly increases the risk of GVHD and reduces the patient's disease-free survival rate.

[0005] The twelve classical HLA loci, HLA-A, B, C, DPA1, DPB1, DQA1, DQB1, DRA, DRB1, DRB3 / 4 / 5, are closely related to hematopoietic stem cells, organ transplant rejection, and prognosis. With the development of DNA typing, the number of known alleles of the above twelve antigens has also increased dramatically. In 2017, the international IPD-IMGT / HLA database included 17,509 alleles, which increased to 38,909 by January 2024. The above-mentioned newly added HLA types cannot be covered by the existing SBT typing kits. In addition, with the increase in the number of HLA alleles, the ambiguity of the HLA SBT typing results has also increased, which has posed a daunting challenge to effective donor-recipient matching and exposed the shortcomings of SBT typing technology. In addition, the first-generation sequencing technology has the disadvantages of complex experimental procedures, high technical requirements for analysts, long detection cycles, high costs, and failure to cover all exon regions, which is not suitable for hospitals to carry out this testing project.

[0006] NGS high-throughput sequencing technology can perform large-scale parallel sequencing and increase the data output of each processing by 1,000 times, reducing costs and processing many samples at the same time. Secondly, NGS uses a cloning method to handle related polymorphisms in heterozygous samples, without the need for additional expensive and time-consuming processes or verification tests to resolve cis-trans ambiguity. Researchers have developed a variety of typing detection kits based on second-generation sequencing technology, such as a method for amplifying short fragments of the major exons of the three HLA loci A, B, and DRB1 and building a library using sample sequence tags; and a method for amplifying long fragments of the full length of the three HLA loci A, B, and C and partial exon regions of the two loci DQB1 and DRB1. Different loci require different PCR amplification systems, and the constructed libraries are tested and compared on two Inmena platforms; and all exons of the eleven HLA loci A, B, C, DPA1, DPB1, DQA1, DQB1, DRB1, and DRB3 / 4 / 5. The method for long-fragment amplification of the sub-region includes 9 sets of amplification primers and a total of 18 primer sequences. The size of the amplified fragment is between 2800bp and 11774bp. The extension time for each cycle of PCR amplification takes 12min to 22min, and a total of 30 cycles are required. In other words, each round of PCR reaction takes 8 hours. The constructed library is sequenced on a third-generation sequencing platform; and a method for long-fragment amplification of the full-length exon regions of the six HLA loci A, B, and C and the three loci DPB1, DQB1, and DRB1. Most of the site typing results displayed can only achieve G group typing results, and fail to achieve sextile typing.

[0007] In summary, the existing HLA typing test kits have the following shortcomings: 1. Due to the defects of the first-generation sequencing technology platform, the HLA typing results are increasingly ambiguous and accurate typing cannot be achieved; 2. The existing detection kits based on the second-generation sequencing technology platform do not have complete exon coverage and cannot achieve accurate typing; 3. The existing detection kits based on the third-generation sequencing technology platform have a long detection cycle and do not cover the HLA-DRA locus. Summary of the invention

[0008] The purpose of the embodiments of the present application is to provide HLA gene amplification primers, a kit and a sequencing library construction method, aiming to solve the problems of low accuracy and long detection cycle in the existing HLA typing detection technology.

[0009] The first aspect of the embodiment of the present application provides HLA gene amplification primers, including the following 16 primer sets:

[0010] Primer set 1: SEQ ID NO.1 and SEQ ID NO.2; Primer set 2: SEQ ID NO.3 and SEQ ID NO.4; Primer set 3: SEQ ID NO.5 and SEQ ID NO.6; Primer set 4: SEQ ID NO.7 and SEQ ID NO.8; Primer set 5: SEQ ID NO.9 and SEQ ID NO.10; Primer set 6: SEQ ID NO.11 and SEQ ID NO.12; Primer set 7: SEQ ID NO.13 and SEQ ID NO.14; Primer set 8: SEQ ID NO.15 and SEQ ID NO.16; Primer set 9: SEQ ID NO.17 and SEQ ID NO.18; Primer set 10: SEQ ID NO.19 and SEQ ID NO.20; Primer set 11: SEQ ID NO.21 and SEQ ID NO.22; Primer set 12: SEQ ID NO.23 and SEQ ID NO.24; Primer set 13: SEQ ID NO.25 and SEQ ID NO. IDNO.26; Primer set 14: SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30 and SEQ IDNO.31; Primer set 15: SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ IDNO.36; Primer set 16: SEQ ID NO.37, SEQ ID NO.38 SEQ ID NO.39, SEQ ID NO.40 and SEQ IDNO.41.

[0011] Preferably, the HLA gene amplification primers further include the following primer set:

[0012] Primer set 17: SEQ ID NO.42 and SEQ ID NO.43; Primer set 18: SEQ ID NO.44 and SEQ ID NO.45; Primer set 19: SEQ ID NO.46 and SEQ ID NO.47; Primer set 20: SEQ ID NO.48 and SEQ ID NO.49; Primer set 21: SEQ ID NO.50 and SEQ ID NO.51; Primer set 22: SEQ ID NO.52 and SEQ ID NO.53.

[0013] A second aspect of an embodiment of the present application provides a kit for HLA gene sequencing, wherein the kit includes the HLA gene amplification primers provided in the first aspect above.

[0014] The third aspect of the embodiment of the present application provides a method for constructing an HLA gene sequencing library, comprising:

[0015] Using the HLA gene amplification primers provided in the first aspect above to perform an amplification reaction on the test sample to obtain an amplification product;

[0016] The amplified product was fragmented, purified by magnetic beads, and the amplified reaction was enriched by adapter ligation to construct an HLA gene sequencing library;

[0017] The HLA gene sequencing library is sorted, purified and quality inspected.

[0018] The embodiment of the present application provides 16 sets of primer sets for multiplex amplification of HLA genes, which can be used for PCR amplification to obtain amplified genes separately, and can be combined for single-tube multiplex PCR amplification, thereby simplifying the experimental process and improving the sensitivity and accuracy of typing. The preparation of libraries using the amplified products of the primer set of the present application can be applied to the detection of alleles of six genomes based on the sequencing technology of the second-generation sequencing platform, and has the advantages of short detection cycle and can meet the accuracy of six-point typing, solving the problems of low accuracy and long detection cycle of existing detection technologies.

[0019] In addition, the embodiments of the present application also provide 22 sets of HLA gene multiplex amplification primer sets, which can further detect more comprehensive alleles of twelve HLA loci, solving the problem that existing detection technologies cover few loci, incomplete exon coverage, and cannot achieve accurate typing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A map of the design positions of the HLA gene amplification primers provided in the embodiments of the present application;

[0021] Figure 2 This is a gel electrophoresis diagram after multiple amplification of six loci provided in the embodiment of the present application;

[0022] Figure 3 The fragment size distribution diagram of the six-locus sequencing library provided in the embodiment of the present application;

[0023] Figure 4 A statistical chart of the amount of data from the six-locus sequencing library provided in the embodiment of the present application;

[0024] Figure 5 A statistical graph of the utilization of six-locus sequencing library data provided in the embodiments of the present application;

[0025] Figure 6 A statistical graph of the length of the fragment analysis of the six-locus sequencing library provided in the embodiment of the present application;

[0026] Figure 7 A statistical graph of coverage depth of six-locus sequencing amplicons provided in an embodiment of the present application;

[0027] Figure 8 A statistical graph of the balance of six-locus sequencing amplicons provided in an embodiment of the present application;

[0028] Fig. 9 A statistical graph of the allele ratios of the six loci provided in the embodiments of the present application;

[0029] Fig.10 A statistical graph of running time for different amounts of analysis data for six loci provided in the embodiments of the present application;

[0030] Fig.11 A statistical graph of coverage depth of different analysis data volumes for the six loci provided in the embodiments of the present application;

[0031] Fig.12 This is a statistical graph of the balance of twelve gene loci sequencing amplicons provided in the embodiments of the present application. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0033] The present application provides HLA gene amplification primers, including the following 16 primer sets:

[0034] Primer set 1: SEQ ID NO.1 and SEQ ID NO.2; Primer set 2: SEQ ID NO.3 and SEQ ID NO.4; Primer set 3: SEQ ID NO.5 and SEQ ID NO.6; Primer set 4: SEQ ID NO.7 and SEQ ID NO.8; Primer set 5: SEQ ID NO.9 and SEQ ID NO.10; Primer set 6: SEQ ID NO.11 and SEQ ID NO.12; Primer set 7: SEQ ID NO.13 and SEQ ID NO.14; Primer set 8: SEQ ID NO.15 and SEQ ID NO.16; Primer set 9: SEQ ID NO.17 and SEQ ID NO.18; Primer set 10: SEQ ID NO.19 and SEQ ID NO.20; Primer set 11: SEQ ID NO.21 and SEQ ID NO.22; Primer set 12: SEQ ID NO.23 and SEQ ID NO.24; Primer set 13: SEQ ID NO.25 and SEQ ID NO. IDNO.26; Primer set 14: SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30 and SEQ IDNO.31; Primer set 15: SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ IDNO.36; Primer set 16: SEQ ID NO.37, SEQ ID NO.38 SEQ ID NO.39, SEQ ID NO.40 and SEQ IDNO.41.

[0035] In the present application example, the relationship between the molar ratios of the working solution concentrations of the primers in each primer set is:

[0036] SEQ ID NO.1 and SEQ ID NO.2 are 1:2; SEQ ID NO.3 and SEQ ID NO.4 are 1:1; SEQ ID NO.5 and SEQ ID NO.6 are 1:4; SEQ ID NO.7 and SEQ ID NO.8 are 3:8; SEQ ID NO.9 and SEQ ID NO.10 are 5:4; SEQ ID NO.11 and SEQ ID NO.12 are 4:5; SEQ ID NO.13 and SEQ ID NO.14 are 1:1; SEQ ID NO.15 and SEQ ID NO.16 are 1:1; SEQ ID NO.17 and SEQ ID NO.18 are 2:3; SEQ ID NO.19 and SEQ ID NO.20 are 1:2; SEQ ID NO.21 and SEQ ID NO.22 are 1:1; SEQ ID NO.23 and SEQ ID NO.24 are 38:63; SEQ ID NO.25 and SEQ ID NO.26 are 5:4; SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO. ID NO.29, SEQ ID NO.30 and SEQ ID NO.31 are 12:12:20:25:12; SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36 are 100:50:100:37:25; SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40 and SEQ ID NO.41 are 6:50:10:25:6.

[0037] In the present application example, the molar ratio of the working solution concentration of each primer set is:

[0038] 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: Primer set 10: Primer set 11: Primer set 12: Primer set 13: Primer set 14: Primer set 15: Primer set 16 = 60: 16: 25: 55: 45: 45: 20: 24: 50: 15: 10: 101: 18: 81: 312: 199.

[0039] The present application also provides another HLA gene amplification primer, including 22 primer sets, that is, based on the above embodiment, it also includes the following primer sets:

[0040] Primer set 17: SEQ ID NO.42 and SEQ ID NO.43; Primer set 18: SEQ ID NO.44 and SEQ ID NO.45; Primer set 19: SEQ ID NO.46 and SEQ ID NO.47; Primer set 20: SEQ ID NO.48 and SEQ ID NO.49; Primer set 21: SEQ ID NO.50 and SEQ ID NO.51; Primer set 22: SEQ ID NO.52 and SEQ ID NO.53.

[0041] In the present application example, the relationship between the molar ratios of the working solution concentrations of the primers in each primer set is:

[0042] SEQ ID NO.42 and SEQ ID NO.43 are 1:1; SEQ ID NO.44 and SEQ ID NO.45 are 1:1; SEQ ID NO.46 and SEQ ID NO.47 are 1:1; SEQ ID NO.48 and SEQ ID NO.49 are 2:1; SEQ ID NO.50 and SEQ ID NO.51 are 1:1; SEQ ID NO.52 and SEQ ID NO.53 are 1:1.

[0043] In the present application example, the molar ratio of the working solution concentration of each primer set is:

[0044] 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: Primer set 10: Primer set 11: Primer set 12: Primer set 13: Primer set 14: Primer set 15: Primer set 16: Primer set 17: Primer set 18: Primer set 19: Primer set 20: Primer set 21: Primer set 22 = 60: 16: 25: 55: 45: 45: 20: 24: 50: 15: 10: 101: 18: 81: 312: 199: 30: 200: 80: 60: 80: 60.

[0045] The present application also provides a kit for HLA gene sequencing, which includes the above-mentioned HLA gene amplification primers.

[0046] It is worth noting that for HLA genotyping, sextile typing is required to determine the amino acids encoded by the HLA gene, and thus the encoded protein, that is, at least all exons of the HLA gene need to be covered, and the existing detection kits do not cover all exon regions due to the limitation of long-fragment amplification, so this application considers the method of short-fragment multiple amplification, although the multiple amplification system of multiple pairs of primers is technically difficult to implement, and the HLA gene family shows a high degree of polymorphism, which poses a challenge to the specificity of primer sequence design. Finally, the innovatively designed kit of this application can not only meet the sextile typing accuracy, but also solve the problem of long-fragment amplification, and also shorten the experimental cycle.

[0047] The present application also provides a method for constructing an HLA gene sequencing library, including:

[0048] Using the above-mentioned HLA gene amplification primers to perform an amplification reaction on the test sample to obtain an amplification product;

[0049] The amplified product was fragmented, purified by magnetic beads, and the amplified reaction was enriched by adapter ligation to construct an HLA gene sequencing library;

[0050] The HLA gene sequencing library is sorted, purified and quality inspected.

[0051] The amplification reaction program was: 95°C×2min; 95°C×30sec, 58~66°C×30sec, 68°C×2min, 25 cycles; 68°C×10min.

[0052] Among them, the adapter ligation enrichment amplification program is: 72℃, 180sec; 98℃, 30sec; 98℃, 15sec, 65℃, 30sec, 72℃, 30sec, 6~9 cycles; 72℃, 300sec.

[0053] The fragmentation procedure is: 55°C, 10~20min.

[0054] In order to make the above scheme more specific, the following specific embodiments are provided.

[0055] Example 1: HLA six loci detection experimental method and result analysis

[0056] This example uses 14 cell line reference samples to study the technical feasibility of the present application for detecting six HLA loci. The specific detection method includes the following steps:

[0057] S1, genomic DNA extraction;

[0058] The 14 cell line reference samples S1~S14 came from the standard laboratory of Shanghai Dishuo Beacon Biotechnology Co., Ltd. They are immortalized cells transformed from human whole blood. The HLA genotyping results have been obtained by the gold standard method of first-generation sequencing. The results are shown in Table 1-2 and can be used for the accuracy evaluation of HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, and HLA-DRB3 / 4 / 5 typing detection kits.

[0059]

[0060]

[0061]

[0062] Use the nucleic acid extraction kit produced by Shanghai Dishuo Beiken Biotechnology Co., Ltd. for extraction. Please strictly follow the instructions for the extraction process. Ensure that the genomic DNA concentration of the 14 cell line reference samples is >10ng / μL and the volume is >20μL. Dilute the genomic DNA with a concentration higher than 50ng / μL. Finally, the genomic DNA of the 14 cell line reference samples is between 10ng / μL and 50ng / μL.

[0063] S2, genomic DNA amplification;

[0064] The genomic DNA was amplified by PCR using the kit of the present application. The components of the kit are shown in Table 3.

[0065]

[0066]

[0067] Multiplex amplification MIX is a commercial reagent, and the supplier is Bao Bio (Cat. No.: RR062A). Other manufacturers such as Novogene (Cat. No.: PM201) and Jinan Bio (E086-01A) can also be used; Primer Mix A is a mixture of 16 primer sets (combination 1 to combination 16), which is used for genotyping detection of six loci; Primer Mix B is a mixture of 22 primer sets (combination 1 to combination 22), which is used for genotyping detection of twelve loci. According to the allele sequence comparison data in the IMGT / HLA database, specific primer sequences are designed in the conserved region of the intron. The primer design positions are as follows: Figure 1 The primers were commissioned to Bioengineering (Shanghai) Co., Ltd. for synthesis. The primer sequences, concentrations, coverage ranges and amplified fragment sizes are shown in Table 4.

[0068]

[0069]

[0070]

[0071]

[0072] The amplification reaction system was prepared using the above detection kit, with a total reaction system of 25 μL and the amount of each component added as shown in Table 5.

[0073]

[0074] After the reaction tubes were tightly capped and centrifuged briefly, PCR amplification reactions were performed on ABI 9700. The amplification reaction program was shown in Table 6. A negative control (NTC) was required for each batch of experiments.

[0075]

[0076] S3, multiplex amplification product quality inspection;

[0077] After the PCR reaction is completed, the PCR reaction product is tested by agarose gel electrophoresis. It is recommended to use 0.8% w / v agarose gel. Figure 2 As shown, the size of the amplified fragment ranged from 700 bp to 2500 bp.

[0078] S4, sequencing library construction;

[0079] The TruePrep DNA Library Prep Kit V2 for Illumina (Novozyme, TD501-02) was used in combination with the TruePrep Index Kit V2 for Illumina (Novozyme, TD202 / TD203) to perform subsequent library construction operations on the multiple PCR amplification products. This application optimizes the use of commercial library construction kits, including amplification product fragmentation, magnetic bead purification, adapter ligation enrichment, sorting and purification, and library fragment quality inspection. The specific operation steps are as follows. This step can also be replaced by other sequencing universal library construction kits, such as: IGT@ Enzyme Plus Library Prep Kit V3 (Aijitaikang, C11112) kits and other similar principle library construction reagents.

[0080] Amplification product fragmentation:

[0081] Calculate the amount of each reagent according to the number of reaction wells. Take out 5×TTBL and thaw it ten minutes in advance. Mix the reagents and prepare the fragmented MIX (MIX does not contain a template) according to Table 7. After the MIX is prepared, place it in a freezing box for use.

[0082]

[0083] Add the required MIX to a new eight-tube strip, adding 23μL to each well. After adding the MIX, apply the sealing film, centrifuge briefly at 2000rpm, and check whether the requirements are met. Use a pipette to draw 2μL of PCR product template into the corresponding well of the fragmented eight-tube strip. Cover with a white silicone pad, centrifuge briefly at 2000rpm, and check whether the requirements are met. Place the eight-tube strip in the PCR instrument, and the reaction program is shown in Table 8. This step is to break the amplicon into a fragment size that can be used for second-generation sequencing.

[0084]

[0085] Magnetic bead purification:

[0086] This step is to interrupt the enzyme digestion reaction process and remove primer dimers and some short fragment products.

[0087] a) Take out the magnetic beads (Novozyme, N411-02) in advance, vortex and mix well, and then return to room temperature, or directly take out the magnetic beads from the refrigerator and vortex for 5 minutes before use.

[0088] b) Take 15 μL of magnetic beads (0.6×) and add them to the fragmentation products in the eight-tube strip. Mix by pipetting. Then let it stand at room temperature for 5 minutes, centrifuge briefly, and then move it to a magnetic stand and let it stand for 4 minutes to fully separate the magnetic beads from the supernatant.

[0089] c) Use a 100μL pipette to gently aspirate the supernatant in the eight-tube strip and discard it. Try to aspirate as much of the supernatant as possible and avoid aspirating the magnetic beads.

[0090] d) Add 100 μL of freshly prepared 80% ethanol to the magnetic plate to rinse the magnetic beads, let it stand for 30 seconds, and then aspirate and discard the ethanol.

[0091] e) Repeat the above washing step d), cover with a white silicone pad, centrifuge briefly, place on a magnetic stand for adsorption, and use a 10μL pipette tip to aspirate and discard the ethanol.

[0092] f) Keep the eight-tube strip on the magnetic rack at all times, open the lid and let it stand until the magnetic beads are completely dry (the surface of the magnetic beads is matte, usually 5 to 10 minutes, the time can be shortened or extended, the specific time is adjusted according to the indoor temperature and humidity to avoid the magnetic beads from drying out).

[0093] g) After the magnetic beads are completely dry, add 15 μL TE (Sanggong, B541019-100) to the eight-tube strip.

[0094] h) Use a pipette to blow and check whether the magnetic beads are fully mixed with TE. Then let it stand on the table for 5 minutes, centrifuge briefly, and then move it to the magnetic stand and let it stand for 2-4 minutes to fully separate the magnetic beads from the supernatant.

[0095] Adapter ligation enrichment:

[0096] Calculate the amount of each reagent according to the number of reaction wells, and prepare the secondary amplification MIX (MIX does not contain a template) according to Table 9. After the MIX is prepared, thaw the required Index Mix at room temperature and place it in a freezing box for later use.

[0097]

[0098] Use a pipette to draw 12 μL of the purified fragmented product into the corresponding wells of the secondary amplification eight-tube strip; draw 2.5 μL of Indexex Mix into the corresponding wells (this step needs to be checked repeatedly). After a short centrifugation at 2000 rpm, check whether the requirements are met. Place the eight-tube strip in the PCR instrument, and the amplification reaction program is shown in Table 10. This step is to add sample labels to different samples so that different samples can be sequenced in parallel.

[0099]

[0100] Sorting and purification:

[0101] This step is to remove long fragment amplification products and sort out fragments in the 400bp~700bp region.

[0102] a) Prepare a 0.2mL or 1.5mL centrifuge tube (depending on the sample volume), pipette 10μL of the secondary amplification product into each reaction well (make sure that the Index Mix of each sample in the mixed sample is different), mix thoroughly, centrifuge briefly, and pipette 100μL for subsequent magnetic bead sorting and purification (if less than 100μL, add nuclease-free water to 100μL).

[0103] b) Take 55 μL of magnetic beads (0.55×) and add them to the above 100 μL mixed solution. Vortex mix on a mixer for at least 10 seconds, then let it stand at room temperature for 5 minutes. Centrifuge briefly and then move to a magnetic rack and let it stand for 5 minutes to fully separate the magnetic beads from the supernatant.

[0104] c) Use a 200μL pipette to gently aspirate the supernatant and transfer it to a new EP tube. Try to aspirate as much of the supernatant as possible and avoid aspirating the magnetic beads. Discard the magnetic beads.

[0105] d) Take 10 μL of magnetic beads (0.1×) and mix thoroughly with the supernatant, then let it stand at room temperature for 5 minutes, and then move it to a magnetic rack and let it stand for 5 minutes to allow the magnetic beads to be fully separated from the supernatant.

[0106] e) Use a 200 μL pipette to gently aspirate the supernatant and discard it, taking care to remove as much of the supernatant as possible and avoid aspirating the magnetic beads.

[0107] f) Add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads by swirling along the wall of the EP tube, let it stand for 2 minutes, and then aspirate and discard the ethanol.

[0108] g) Repeat the above washing step f), cover with a white silicone pad, centrifuge briefly, place on a magnetic stand for adsorption, and discard the ethanol.

[0109] h) Keep the EP tube on the magnetic rack at all times, open the tube cover and let it stand until the magnetic beads are completely dry (the surface of the magnetic beads is matte, usually about 5 minutes, the time can be shortened or extended, the specific time is adjusted according to the indoor temperature and humidity, and the magnetic beads are completely dry).

[0110] i) Keep the magnetic plate on the magnetic stand and add 43 μL 1×TE to the magnetic plate. If the number of samples is less than 10, add TE in proportion (for example, 20 μL TE is used for 5 samples).

[0111] j) Use a vortex mixer to mix for at least 10 seconds or use a pipette to pipette to check whether the magnetic beads are fully mixed with TE. Then let it stand on the table for 5 minutes, and then move it to the magnetic stand and let it stand for 2 minutes to allow the magnetic beads to be fully separated from the supernatant.

[0112] k) Use a pipette to pipette 40 μL of supernatant into new EP tubes. If the number of samples is less than 10, pipette 0.8× the elution volume of supernatant.

[0113] Library fragment quality inspection:

[0114] The nucleic acid concentration and fragment size of the final library were determined using the Qubit dsDNA HS (Thermo Fisher, Q32854) detection kit and High Sensitivity D1000 Reagents (Agilent, 5067-5585). Figure 3 shown.

[0115] S5, sequencing library and result analysis;

[0116] After the constructed library was loaded on the second-generation sequencing platform Illumina Novaseq, the amount of data from 14 cell line reference samples was as follows: Figure 4 As shown in the figure, the off-machine data was analyzed using the open source analysis process HLA-HD (Version 1.7.0) and the self-developed analysis method. After the off-machine data of each reference sample was deduplicated and low-quality data was removed, the data utilization rate of each sample was as follows: Figure 5As shown; This application analyzes the available data for each library, including the length of library fragment analysis ( Figure 6 )、16 groups of amplicon coverage depth ( Figure 7 )、16 groups of amplicon balance( Figure 8 ) and allele proportion ( Fig. 9 ). The average length of the library fragment analysis is 282bp, including the length of the adapter, which is equivalent to the fragment size detection result; the average coverage depth of the 16 groups of amplicons is between 1767 and 5743, the average amplicon balance is between 0.4 and 1.3, the allele ratio is between 20% and 80%, and the smallest allele ratio is greater than 20%. At the same time, this application compares the running time and coverage depth of the self-developed analysis method under different analysis data volumes (100MB, 50MB, 20MB), and finds that the lower the data volume, the shorter the analysis time ( Fig.10 ), the lower the coverage depth ( Fig.11 ). The results of the six-locus typing of the second-generation sequencing of the cell line reference samples are shown in Table 11. The results showed that the quartile analysis results of the second-generation sequencing of the self-developed analysis method for 14 cell line reference samples were consistent with the first-generation sequencing results, with an accuracy rate of 100%, and the detection results of the method of this application can obtain six-quartile accuracy typing results, which are more accurate than the first-generation results. This application subsequently tested more than 1,000 clinical samples, and the test results were consistent with the first-generation sequencing test results (the specific test comparison results are not shown due to the large amount of data). These analysis data show that the HLA typing kit detection method established by this application is accurate and reliable.

[0117]

[0118]

[0119]

[0120] Example 2: Experimental method and result analysis of detection of twelve HLA loci

[0121] This example still uses the above 14 cell line reference samples to study the technical feasibility of detecting the twelve HLA loci in this application. The specific detection method includes the following steps:

[0122] The detection process is basically the same as the six-locus detection method, from genome extraction, PCR amplification, library sequencing and data analysis. The difference lies in the twelve-locus PCR amplification reaction system. The reaction system is prepared as follows:

[0123]

[0124] After the PCR reaction is completed, the PCR reaction product is quality checked by agarose gel electrophoresis. It is recommended to use 0.8% w / v agarose gel. The amplicon fragment size is between 700 bp and 2500 bp.

[0125] After the second-generation sequencing data was downloaded, the open source analysis process HLA-HD (Version 1.7.0) and self-developed analysis methods were used for analysis. This application analyzed the available data of each library, including 22 groups of amplicon balance ( Fig.12 ), the mean values ​​of the balance of the 22 groups of amplicons are between 0.4 and 1.3, which can be adjusted and optimized by adjusting the ratio of multiple amplification primers; the allele ratio is between 20% and 80%, and the minimum allele ratio is greater than 20%. The typing results of the other twelve loci of the second-generation sequencing of the cell line reference samples are shown in Table 13, of which the typing results of six loci are consistent with the detection results of Example 1, so they are not shown. The results showed that the quartile analysis results of the second-generation sequencing of 14 cell line reference samples by the self-developed analysis method were consistent with the first-generation sequencing results, with an accuracy rate of 100%, and the detection results of the method of this application can obtain typing results with six quartile accuracy, which is more accurate than the first-generation results.

[0126]

[0127]

[0128] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. HLA gene amplification primer, characterized in that: Includes the following 16 primer sets: Primer set 1: SEQ ID NO.1 and SEQ ID NO.2; Primer set 2: SEQ ID NO.3 and SEQ ID NO.4; Primer set 3: SEQ ID NO.5 and SEQ ID NO.6; Primer set 4: SEQ ID NO.7 and SEQ ID NO.8; Primer set 5: SEQ ID NO.9 and SEQ ID NO.10; Primer set 6: SEQ ID NO.11 and SEQ ID NO.12; Primer set 7: SEQ ID NO.13 and SEQ ID NO.14; Primer set 8: SEQ ID NO.15 and SEQ ID NO.16; Primer set 9: SEQ ID NO.17 and SEQ ID NO.18; Primer set 10: SEQ ID NO.19 and SEQ ID NO.20; Primer set 11: SEQ ID NO.21 and SEQ ID NO.22; Primer set 12: SEQ ID NO.23 and SEQ ID NO.24; Primer set 13: SEQ ID NO.25 and SEQ IDNO.26; Primer set 14: SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30 and SEQ IDNO.31; Primer set 15: SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ IDNO.36; Primer set 16: SEQ ID NO.37, SEQ ID NO.38 SEQ ID NO.39, SEQ ID NO.40 and SEQ IDNO.

41.

2. The HLA gene amplification primer according to claim 1, characterized in that: The relationship between the molar ratios of the working solution concentrations of the primers in each primer set is: SEQ ID NO.1 and SEQ ID NO.2 are 1:2; SEQ ID NO.3 and SEQ ID NO.4 are 1:1; SEQ ID NO.5 and SEQ ID NO.6 are 1:4; SEQ ID NO.7 and SEQ ID NO.8 are 3:8; SEQ ID NO.9 and SEQ ID NO.10 are 5:4; SEQ ID NO.11 and SEQ ID NO.12 are 4:5; SEQ ID NO.13 and SEQ ID NO.14 are 1:1; SEQ ID NO.15 and SEQ ID NO.16 are 1:1; SEQ ID NO.17 and SEQ ID NO.18 are 2:3; SEQ ID NO.19 and SEQ ID NO.20 are 1:2; SEQ ID NO.21 and SEQ ID NO.22 are 1:1; SEQ ID NO.23 and SEQ ID NO.24 are 38:63; SEQ ID NO.25 and SEQ ID NO.26 are 5:4; SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30 and SEQ ID NO.31 are 12:12:20:25:12; SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36 are 100:50:100:37:25; SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40 and SEQ ID NO.41 are 6:50:10:25:

6.

3. The HLA gene amplification primer according to claim 1, characterized in that: The molar ratio of the working solution concentration 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: Primer set 10: Primer set 11: Primer set 12: Primer set 13: Primer set 14: Primer set 15: Primer set 16 = 60: 16: 25: 55: 45: 45: 20: 24: 50: 15: 10: 101: 18: 81：312：199。 4. The HLA gene amplification primer according to claim 1, characterized in that: The following primer sets are also included: Primer set 17: SEQ ID NO.42 and SEQ ID NO.43; Primer set 18: SEQ ID NO.44 and SEQ ID NO.45; Primer set 19: SEQ ID NO.46 and SEQ ID NO.47; Primer set 20: SEQ ID NO.48 and SEQ ID NO.49; Primer set 21: SEQ ID NO.50 and SEQ ID NO.51; Primer set 22: SEQ ID NO.52 and SEQ ID NO.

53.

5. The HLA gene amplification primer according to claim 4, characterized in that: The relationship between the molar ratios of the working solution concentrations of the primers in each primer set is: SEQ ID NO.42 and SEQ ID NO.43 are 1:1; SEQ ID NO.44 and SEQ ID NO.45 are 1:1; SEQ ID NO.46 and SEQ ID NO.47 are 1:1; SEQ ID NO.48 and SEQ ID NO.49 are 2:1; SEQ ID NO.50 and SEQ ID NO.51 are 1:1; SEQ ID NO.52 and SEQ ID NO.53 are 1:

1.

6. The HLA gene amplification primer according to claim 4, characterized in that: The molar ratio of the working solution concentration 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: Primer set 10: Primer set 11: Primer set 12: Primer set 13: Primer set 14: Primer set 15: Primer set 16: Primer set 17: Primer set 18: Primer set 19: Primer set 20: Primer set 21: Primer set 22 = 60: 16: 25: 55: 45: 45: 20: 24: 50: 15: 10: 101: 18: 81: 312: 199: 30: 200: 80: 60: 80:

60.

7. A kit for HLA gene sequencing, characterized in that: The kit comprises the HLA gene amplification primers according to any one of claims 1-6.

8. A method for constructing an HLA gene sequencing library, characterized in that: include: Using the HLA gene amplification primers described in any one of claims 1 to 6 to perform an amplification reaction on the test sample to obtain an amplification product; The amplified product was fragmented, purified by magnetic beads, and the amplified reaction was enriched by adapter ligation to construct an HLA gene sequencing library; The HLA gene sequencing library is sorted, purified and quality inspected.

9. The method for constructing an HLA gene sequencing library according to claim 8, characterized in that: The amplification reaction program was: 95°C×2 min; 95°C×30 sec, 58~66°C×30 sec, 68°C×2 min, 25 cycles; 68°C×10 min.

10. The method for constructing an HLA gene sequencing library according to claim 8, characterized in that: The adapter ligation enrichment amplification program was: 72°C, 180 sec; 98°C, 30 sec; 98°C, 15 sec, 65°C, 30 sec, 72°C, 30 sec, 6 to 9 cycles; 72°C, 300 sec.

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