Integrated genotyping method for multiple blood cell antigens related to selective blood transfusion and application of integrated genotyping method

Abstract

The invention relates to the field of medical detection, in particular to an integrated genetic typing method for multiple blood cell antigens related to selective blood transfusion and application of the integrated genetic typing method. The genetic typing method is an integrated typing method which is based on a DNA sequence and relates to a plurality of red blood cells, blood platelets and leukocyte antigen coding genes related to blood transfusion, the occurrence rate of heteroantigen sensitization caused by homoimmunization can be reduced, precise blood transfusion is promoted, blood type genes comprise ABO, RhD, RhCE, HPA (ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109 and GP9), HLA-A, B, C,-DRB1,-DQB1, ABO, FUT1, FUT2 and the like, and the blood type genes are used for preparing the blood transfusion kit. More than tens of thousands of alleles can be covered; in addition, the constructed platform can be used for constructing a second-generation sequencing library and also can be used for constructing a third-generation sequencing library subsequently, an excellent recreation and reproduction platform is provided for haplotype confirmation of multi-site variation in blood group genes in the future, and the method has important significance for accurate blood transfusion in the future.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular, to an integrated gene typing method for multiple blood cell antigens related to selective blood transfusion and its application. Background Art

[0002] There are 362 blood group antigens on the surface of human red blood cells, which are clustered in 45 blood group systems. The surface of human platelets has: ABO and Rh blood group system antigens, platelet-specific antigens (human plateletantigen, HPA), leukocyte antigens (human leukocyte antigen, HLA) and CD36 antigens. There are 35 types of HPA, 19252 types of HLA-A, -B and -C proteins, and 3917 types of HLA-DRB1 and -DQB1 proteins. HLA is also expressed in plasma. In clinical blood transfusion, routine red blood cell transfusion only cross-matches the red blood cell ABO and RhD blood group antigens; routine platelet and plasma transfusions use ABO and RhD homotype transfusions, but do not match antigens such as HLA, HPA and CD36 on the surface of platelets. Under the conventional blood transfusion matching strategy, the probability of transfusing non-self antigens is high. Studies have shown that blood type antigens detected based on DNA sequencing are more extensive and accurate, and blood type gene matching transfusion strategies can improve the effectiveness and safety of blood transfusions. This is especially important for patients who need repeated blood use, difficult matching, have a history of blood transfusion or pregnancy, and have undergone organ transplantation and ineffective transfusions.

[0003] However, in current clinical transfusion, conventional red blood cell, platelet and plasma transfusion only focuses on the matching of red blood cell ABO and RhD blood type antigens, and does not match HPA, HLA and CD36. Although serological methods are simple, easy and cheap, they cannot avoid the input and sensitization of allogeneic antigens. For patients who already have a history of sensitization (blood transfusion history, pregnancy history, organ transplantation, etc.), need repeated blood transfusions, platelet transfusion ineffectiveness, autoimmune diseases, cancer, etc., this conventional blood matching strategy will bring very serious transfusion complications, which is not only not conducive to the treatment of the primary disease, but also may be life-threatening.

[0004] In the prior art, CN113215237A discloses a simultaneous detection kit and method for HPA and HLA antigen systems, which uses a probe capture method to perform typing tests on HPA and HLA-A, -B, and -C. In order to cover the detection area as much as possible, the scheme designed a large number of probes and continuously optimized them, and finally selected up to 498 probes. In addition, the scheme requires mechanical interruption and recapture of gDNA extracted from each sample. The operation process is complicated and the operation cost is expensive. It is not suitable for typing tests of a large number of samples, and the patent only detects HPA and HLA-I genes. CN114410758A discloses a method and kit for detecting HPA genotypes with high efficiency and specificity. The scheme uses an allele-specific PCR method to detect HPA genotypes. Each sample needs to be amplified in multiple reaction tubes using multiple sets of primers. The operation is complicated, and only HPA polymorphic sites 1 to 6, 15, and 21 are typed. CN111218514A discloses a method and reagent kit for determining the full-length sequence of the ABO gene based on the second-generation sequencing technology. The scheme uses the transposase method to build a library and perform NGS sequencing on the amplified ABO gene. The operation process is complicated, and the transposase interruption has a certain preference. This patent only performs typing detection on the ABO gene. In summary, there is currently no gene typing detection method that covers blood type antigens comprehensively, is simple to operate, and has a high accuracy rate. Summary of the invention

[0005] In order to overcome the limitations of conventional blood transfusion matching and the shortcomings of the prior art, the present application provides a genetic typing detection method for red blood cell antigens, platelet antigens and leukocyte antigens related to selective blood transfusion, which has the advantages of full coverage, simple operation, high throughput and high accuracy. Specifically, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a primer set for selective transfusion-related genotyping detection, wherein the primer set comprises the sequences shown in SEQ ID NO.1-82.

[0007] The second aspect of the present invention provides an integrated genotyping method for multiple blood cell antigens related to selective blood transfusion, the method comprising:

[0008] (4) using multiplex PCR amplification to achieve enrichment of the coding regions of multiple selective transfusion-related antigens in one tube;

[0009] (5) constructing a second-generation or third-generation sequencing library using the enriched nucleic acid fragments;

[0010] (6) Genotyping of relevant blood cell antigen genes.

[0011] Among them, the encoding genes of the selective transfusion-related antigens include ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109, GP9, HLA-A, -B, -C, -DRB1, -DQB1, ABO, FUT1, FUT2, RhD, RhCE and CD36.

[0012] In one embodiment, the primer mixture used in the multiplex PCR amplification in step (1) is a long fragment amplification reagent, and the amplification length range is 1kb-35kb, preferably 2kb-10kb, and further preferably 3kb-7kb; the long fragment amplification reagent can amplify the target fragment with a multiplicity range of 1-500 (number of PCR products), preferably 10-400, and further optimized to 100-200.

[0013] In a preferred embodiment, the primer is a sequence as shown in SEQ ID NO.1-82.

[0014] In one embodiment, the method specifically comprises the following steps:

[0015] (1) using the nucleic acid extracted from the sample as a template, preparing an amplification system with primer sets having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.82, respectively, and performing a first amplification reaction to obtain a first amplification product;

[0016] (2) fragmenting the first amplified product, repairing the ends, and adding an A tail;

[0017] (3) purifying the product of step (2) after linking;

[0018] (4) Sequencing the purified product to obtain the genotyping results of the relevant blood cell antigens.

[0019] It has good amplification efficiency and specificity, and there will be no mutual interference between primers during multiple primer amplification. By adjusting the ratio of amplification primers, the focus area of ​​each gene can achieve effective multiple coverage to ensure the accuracy of genotyping.

[0020] Compared with the prior art, the present invention has achieved the following beneficial technical effects:

[0021] 1. The genotyping method described in the present invention is an integrated typing method based on DNA sequences involving multiple red blood cell, platelet and leukocyte antigen encoding genes related to blood transfusion, which can reduce the incidence of alloimmunization-induced xenoantigen sensitization and promote accurate blood transfusion; support blood transfusion matching for complex clinical cases: suitable for complex cases requiring highly matched blood products, such as fetal neonatal immune thrombocytopenia (FNAIT) and immune thrombocytopenia patients in high-risk pregnancies;

[0022] 2. The blood type genes covered by this patent research and development include: ABO, RhD, RhCE, HPA (ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109, GP9), HLA-A, -B, -C, -DRB1, -DQB1, ABO, FUT1, FUT2, etc., covering tens of thousands of alleles;

[0023] 3. The method of the present invention can also take into account the advantages of second-generation and third-generation sequencing: the constructed platform can subsequently construct both second-generation sequencing libraries and third-generation sequencing libraries, providing an excellent re-creation and reproduction platform for the haplotype confirmation of multi-site variations in blood type genes in the future, which is of great significance for future precision blood transfusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 This is a diagram of the sequencing results of Example 3 of the present invention. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0027] The reagents used in the examples are as follows:

[0028] DNA extraction kit: TIANamp Blood DNA Kit, Tiangen, DP-348

[0029] PCR enzymes: 2x Phanta Flash Master Mix, Vazyme, P510

[0030] NGS library preparation: TIANSeq DirectFastLibrary Kit, Tiangen, NG101

[0031] Example 1 Determination of important blood group antigens related to selective blood transfusion and screening of primers

[0032] Based on the limitations of conventional blood transfusion matching strategies and domestic and foreign literature research, the important blood type antigens related to selective blood transfusion were identified, and then the coding genes and heat mutation sites of the antigens involved were clarified.

[0033] Through the previous retrospective study, the important blood cell antigen genes (18 genes) were determined to be: ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109, GP9, HLA-A, -B, -C, -DRB1, -DQB1, ABO, FUT1, FUT2, RhD, RhCE and CD36. Long fragment amplification primers were designed for the above areas of concern, including a total of 5 primer sets:

[0034] (A) The amplification region of the HPA primer set is shown, including:

[0035] 1) Exons exon1, exon2, exon3 of the GP9 gene on chromosome 3 of the human genome;

[0036] 2) Exons exon13, exon14, exon17, exon18, exon19, exon20, exon21, exon22, exon23, exon28, and exon29 of the ITGA2 gene on chromosome 5 of the human genome;

[0037] 3) Exons exon16, exon17, exon18, exon19, exon20 of CD109 gene on chromosome 6 of the human genome;

[0038] 4) Exons exon1 and exon2 of the GP1BA gene on chromosome 17 of the human genome;

[0039] 5) Exons exon2, exon3, exon4, exon5, exon6, exon7, exon8, exon9, exon10, exon11, exon13, exon14, exon15, exon16, exon17, exon18, exon19, exon20, exon21, exon22, exon23, exon24, exon25, exon26, exon27 of the ITGA2B gene on chromosome 17 of the human genome;

[0040] 6) Exons exon2, exon3, exon4, exon5, exon6, exon9, exon10, exon11, exon12 of the ITGB3 gene on chromosome 17 of the human genome;

[0041] 7) Exons exon1 and exon2 of the GP1BB gene on chromosome 22 of the human genome.

[0042] (ii) The amplification region of the CD36 primer set is shown, including:

[0043] 1) The 5'UTR region of the CD36 gene on chromosome 7 of the human genome;

[0044] 2) exon 2 and exon 3 of the CD36 gene on chromosome 7 of the human genome;

[0045] 3) exon 4 and exon 5 of the CD36 gene on chromosome 7 of the human genome;

[0046] 4) exon6, exon7, exon8 of the CD36 gene on chromosome 7 of the human genome;

[0047] 5) exon9, exon10, exon11, exon12, exon13, exon14 of the CD36 gene on chromosome 7 of the human genome;

[0048] 6) Exon 15 and 3'UTR region of CD36 gene on chromosome 7 of human genome.

[0049] (III) The amplification region of the ABOFUT primer set is shown, including:

[0050] 1) ABO gene exon1 on chromosome 9 of the human genome;

[0051] 2) ABO genes exon2, exon3, exon4, exon5, exon6 on chromosome 9 of the human genome;

[0052] 3) ABO gene exon7 on chromosome 9 of the human genome;

[0053] 4) exon 4 of the FUT1 gene on chromosome 19 of the human genome;

[0054] 5) Exon 3 of the FUT2 gene on chromosome 19 of the human genome.

[0055] (IV) The amplification region of the Rh primer set is shown as follows, including:

[0056] 1) 5'UTR region of the RHD gene on chromosome 1 of the human genome;

[0057] 2) RHD gene exon1 on chromosome 1 of the human genome;

[0058] 3) RHCE gene exon1 on chromosome 1 of the human genome;

[0059] 4) RHD gene and RHCE gene exon2 on chromosome 1 of the human genome;

[0060] 5) RHD gene and RHCE gene exon3 on chromosome 1 of the human genome;

[0061] 6) RHD gene and RHCE gene exon4, exon5, exon6 on chromosome 1 of the human genome;

[0062] 7) RHD gene and RHCE gene exon7 on chromosome 1 of the human genome;

[0063] 8) RHD gene and RHCE gene exon8 on chromosome 1 of the human genome;

[0064] 9) RHD gene and RHCE gene exon9 on chromosome 1 of the human genome;

[0065] 10) RHD gene exon10 on chromosome 1 of the human genome;

[0066] 11) RHCE gene exon10 on chromosome 1 of the human genome.

[0067] (V) HLA primer set, including:

[0068] 1) HLA-A gene exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8 on chromosome 6 of the human genome;

[0069] 2) HLA-B gene exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8 on chromosome 6 of the human genome;

[0070] 3) HLA-C gene exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8 on chromosome 6 of the human genome;

[0071] 4) HLA-DQB1 gene exon1, exon2, exon3, exon4, exon5, exon6 on chromosome 6 of the human genome;

[0072] 5) HLA-DRB1 gene exon 1 on chromosome 6 of the human genome;

[0073] 6) HLA-DRB1 gene exon2, exon3, exon4, exon5, exon6 on chromosome 6 on the human genome.

[0074] Primer screening is the key to ensuring efficient amplification, especially when the number of amplified fragments is large. A scientific screening process is essential. When optimizing primer pairs, it is necessary to consider multiple aspects such as primer sequence and PCR reaction system. To this end, we designed and screened the primers for the above gene loci as follows:

[0075] 1. Screening of primer design

[0076] 1) Design candidate primers using dedicated primer design software (such as NCBI Primer-BLAST).

[0077] 2) Use BLAST comparison to exclude nonspecific amplification. Optimize the uniqueness of primer target sequences for complex genomic regions.

[0078] 2. Initial screening: amplification performance of a single primer pair

[0079] 1) Experimental verification: Test each primer pair individually and perform PCR experiments using samples (blood donor samples that have signed informed consent); optimize amplification conditions (such as annealing temperature and number of cycles) to evaluate amplification efficiency and specificity.

[0080] 2) Screening criteria: The target product has a clear single band, amplification efficiency> 90%, and no non-specific bands or primer dimers.

[0081] After the initial round of screening, about 75% of the primer pairs passed the test and entered the next round of screening.

[0082] 3. Second round of screening: compatibility between primer pairs

[0083] 1) Performance evaluation of primer mixing: Mix the primers that have undergone preliminary screening and group them according to fragment size; detect whether there is primer competition or interference in PCR.

[0084] 2) Screening basis: All target product bands in the electrophoresis results are clear and uniform in intensity; there are no primer dimer bands; product specificity is verified by melting curve (such as qPCR).

[0085] After the second round of screening, about 80% of the primer pairs with good performance were retained for the next round of screening.

[0086] 4. Third round of screening: coverage and uniformity of amplified fragments

[0087] 1) Fragment uniformity test: Amplify different templates (such as mixed samples or standard samples) and verify the coverage of multiple amplified fragments. Ensure that all fragments are reliably amplified and there is no significant unevenness in strength.

[0088] 2) Quantitative evaluation: The amount of amplified product was measured by fluorescent quantitative PCR, and the ΔCt value was calculated to determine whether the relative efficiency of fragment amplification was consistent. High-throughput sequencing was used to verify the final product to confirm that all target regions were evenly covered and there was no significant amplification bias.

[0089] In the third round of screening, about 90% of the primers were able to maintain good amplification effects and entered the final optimization.

[0090] 5. Final Optimization: Dynamic Adjustment of Primer Sets

[0091] 1) Primer concentration optimization: According to the fragment amplification efficiency, adjust the concentration of each pair of primers (e.g., appropriately reduce the concentration of efficient primers).

[0092] 2) Multiple rounds of dynamic optimization: Combine all primers, perform a whole set of multiplex PCR, and observe product consistency (ensure the consistency of amplified products under different instruments, different experimental batches, and different operators. Each product can obtain similar brightness and clear bands in the electrophoresis detection experiment, and its sequencing results are consistent). Fine-tune or redesign primers with poor performance based on the results.

[0093] 3) Stability test: Repeat the experiment under different experimental conditions (such as gradient annealing temperature) to ensure the stability of the primer combination (through multiple rounds of experiments, the final primer set showed high stability, the amplification product band intensity was uniform, and the sequence consistency was shown in the high-throughput sequencing experiment, meeting the requirements of high-quality multiplex PCR experiments).

[0094] 6. High-throughput sequencing verification

[0095] 1) Final validation of the primer set: Use next-generation sequencing (NGS) to assess the coverage, specificity, and uniformity of the amplified fragments. Troubleshoot off-target amplification or insufficient coverage of the primers.

[0096] 2) Data analysis: Compare the amplified product sequence with the reference genome to evaluate the integrity of the amplified region. Adjust the primer set optimization according to the sequencing depth to ensure amplification uniformity.

[0097] Through the above scientific and rigorous screening process, the primer set finally obtained meets the following requirements: all target fragments are amplified specifically and evenly; the primer set has good compatibility, efficient and accurate amplification performance, and is suitable for high-throughput and standardized operations; it is highly stable under different experimental conditions and can be used to analyze complex samples and large-scale experiments.

[0098] The detailed information of the primer set finally obtained above is shown in Table 1:

[0099] Table 1 Amplification primer set information

[0100]

[0101]

[0102]

[0103] Implementation Case 2: Using the present invention to construct an NGS sequencing library

[0104] Human whole blood genomic DNA was extracted using a commercial kit (TIANamp Blood DNA Kit, Tiangen, DP-348) and qubit quantification was performed

[0105] 1. Long DNA fragment amplification

[0106] Long fragment DNA amplification system:

[0107]

[0108] Amplification Procedure:

[0109]

[0110]

[0111] After amplification, take 5 μL and perform quality control by 1% agarose gel electrophoresis. If the amplified product is between 4 kb and 8 kb, the quality control is qualified. Figure 1 As shown, after the quality control is qualified, 4 μL is taken for subsequent fragmentation.

[0112] 2. Fragmentation and end repair, filling and A tailing (TIANSeqDirectFastLibraryKit, Tiangen, NG101)

[0113] Experimental system:

[0114]

[0115] Reaction procedure:

[0116]

[0117] 3. Connector connection

[0118] Connector connection system:

[0119]

[0120]

[0121] Connector connection procedure:

[0122]

[0123] 4. Purification

[0124] Purification was performed using 0.8× XP beads, and the purified product was eluted with 26 μL DB.

[0125] 5. Sequencing Primer Amplification

[0126] Sequencing primer (P5, P7 adapter primer) amplification system:

[0127]

[0128] Sequencing primer amplification procedure:

[0129]

[0130] 6. Magnetic Bead Purification

[0131] Purification was performed using 0.8× XP beads, and the purified product was eluted with 26 μL DB and quantified using qubit.

[0132] 7. Sequencing

[0133] The double-end PE150 sequencing was performed using an Illumina sequencer to analyze the sequencing results. The sequencing depth results of the test samples at the variant sites within the target amplified fragment are shown in Table 2:

[0134] Table 2 Sequencing depth of target gene amplified fragments of test samples

[0135]

[0136]

[0137]

[0138] Test sample blood type genotyping results (Table 3):

[0139] Table 3 Test sample related blood type genotyping results antigen

[0140]

[0141]

[0142] It can be seen from this implementation case that the method of the present invention can be used to complete the construction of the second-generation sequencing library, and the sequencing results cover the focus areas of 18 genes of human selective transfusion-related antigens.

[0143] Example 3: Construction of Nanopore sequencing library

[0144] Human whole blood genomic DNA was extracted using a commercial kit (Tiangen DNA Extraction Kit) and subjected to qubit quantification 1 and long-fragment DNA amplification

[0145] The amplification system is as follows:

[0146]

[0147]

[0148] The amplification procedure is as follows:

[0149]

[0150] After amplification, add 37.5 μL of DB to make up to 50 μL, and add 40 μL of XPbeads for purification. After purification, elute with 20 μL of DB, take 5 μL of the eluted product and perform quality control by 1% agarose gel electrophoresis. After passing the quality control, take 500 ng for subsequent library construction.

[0151] 2. End repair and A tail addition

[0152] The experimental system is as follows:

[0153]

[0154] The end repair and A-tailing reaction procedure is as follows:

[0155]

[0156] 3. Native barcode connector connection

[0157] The connection system is as follows:

[0158]

[0159]

[0160] The connection procedure is as follows:

[0161]

[0162] After the ligation reaction is completed, the samples with different barcodes are mixed together for subsequent purification.

[0163] 4. Purification

[0164] Purification was performed using 0.4× XP beads, and the purified product was eluted with 35 μL DB.

[0165] 5. Adaptor connector connection

[0166] The connection system is as follows:

[0167]

[0168] The connection procedure is as follows:

[0169]

[0170] 6. Purification

[0171] Purification was performed using 0.4×XP beads and washing was performed using the Long Fragment Buffer (LFB) provided with the Nanopore kit. The purified product was eluted with 15 μL DB and quantified by qubit.

[0172] 7. Sequencing

[0173] 20 fmol of the purified product was taken for nanopore sequencing. Comparison of the second-generation sequencing results of test sample 3 and the third-generation Nanopore sequencing results of test sample 3 (Table 4):

[0174] Table 4 Comparison of blood type genotyping results of test sample 3 obtained by two sequencing methods

[0175]

[0176]

[0177] The sequencing results of Example 3 show that the method of the present invention can complete the construction and sequencing of the library of the third-generation sequencing. The third-generation nanopore sequencing results of the test sample 3 are consistent with the second-generation sequencing results ( Figure 1 ). In gene analysis, the second generation sequencing (NGS) has difficulty in assembling haplotypes due to its short read length, which may produce ambiguous results, especially in areas of high polymorphism and complex structural variation. However, the third generation sequencing (such as Nanopore) can directly cover the entire gene region by virtue of its long read length advantage, accurately analyze whether the variation is located on the same chromosome, and provide clear information for haplotype assembly. Therefore, the technology of the present invention can not only accurately identify the variation site on the basis of the third generation sequencing, but also identify the haplotype of the variation site, and can accurately perform typing judgment on complex genes.

[0178] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A primer set for detecting genotyping related to selective blood transfusion, characterized in that: The primer set includes the sequences shown as SEQ ID NO.1-82.

2. An integrated genotyping method for multiple blood cell antigens related to selective blood transfusion, characterized in that: The method comprises: (1) Enrichment of the coding regions of multiple transfusion-related antigens in one tube using multiplex PCR amplification; (2) constructing a second-generation or third-generation sequencing library using the enriched nucleic acid fragments; (3) Genotyping of relevant blood cell antigen genes.

3. The method according to claim 2, characterized in that The encoding genes of the selective transfusion-related antigens include ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109, GP9, HLA-A, -B, -C, -DRB1, -DQB1, ABO, FUT1, FUT2, RhD, RhCE and CD36.

4. The method according to claim 2, characterized in that The primer mixture used in the multiplex PCR amplification in step (1) is a long fragment amplification reagent, and the amplification length range is 1kb-35kb, preferably 2kb-10kb, and further preferably 3kb-7kb; the long fragment amplification reagent can amplify the target fragment with a multiplicity range of 1-500 (number of PCR products), preferably 10-400, and further optimized to 100-200.

5. The method according to any one of claims 2 to 4, characterized in that: The primers are sequences shown in SEQ ID NO. 1-82.

6. The method according to any one of claims 2 to 4, characterized in that: The method specifically comprises the following steps: (1) using the nucleic acid extracted from the sample as a template, preparing an amplification system with primer sets having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.82, respectively, and performing a first amplification reaction to obtain a first amplification product; (2) fragmenting the first amplified product, repairing the ends, and adding an A tail; (3) purifying the product of step (2) after linking; (4) Sequencing the purified product to obtain the genotyping results of the relevant blood cell antigens.

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