Method for integrated genotyping of multiple blood cell antigens related to selective blood transfusion and application thereof

Abstract

The present application relates to the field of medical detection, and in particular, the present application relates to a kind of integrated genotyping method of multiple blood cell antigens related to selective blood transfusion and application thereof.The genotyping method described in the present application is an integrated typing method based on DNA sequence related to multiple red blood cell, platelet and white blood cell antigen coding genes related to blood transfusion, which can reduce the incidence of xenoantigen sensitization caused by alloimmunization, promote precision blood transfusion, and cover blood group genes including ABO, RhD, RhCE, HPA (ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109, GP9), HLA-A, -B, -C, -DRB1, -DQB1, ABO, FUT1, FUT2, etc.The covered alleles can reach tens of thousands.Moreover, after the platform built in the present application, second-generation sequencing library construction and third-generation sequencing library construction can be carried out, which provides an excellent re-creation and reproduction platform for future haplotype confirmation of multiple site variations in blood group genes, and has important significance for future precision blood transfusion.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical detection, in particular, the present application relates to a one-step genotyping method for multiple blood cell antigens related to selective blood transfusion and application thereof. BACKGROUND

[0002] There are 362 blood group antigens on the surface of human red blood cells, which are gathered in 45 blood group systems. Human platelets have ABO and Rh blood group system antigens, human platelet antigen (HPA), human leukocyte antigen (HLA) and CD36 antigen on their surface. There are 35 kinds of HPA, 19252 kinds of HLA-A, -B and -C proteins, and 3917 kinds of HLA-DRB1 and -DQB1 proteins. There is also HLA expression in plasma. In clinical blood transfusion, only red blood cell ABO and RhD blood group antigens are matched for routine red blood cell transfusion; ABO and RhD homologous transfusion is used for routine platelet and plasma transfusion, and the platelet surface HLA, HPA and CD36 antigens are not matched. Under the strategy of routine blood transfusion matching, the probability of inputting non-self antigens is high. Studies have shown that blood group antigens detected based on DNA sequencing are more extensive and accurate, and blood group gene matching transfusion strategy can improve the effectiveness and safety of blood transfusion, which is of great significance for patients who need repeated blood transfusion, difficult matching, have a history of blood transfusion or pregnancy, organ transplantation and ineffective transfusion, etc.

[0003] However, in current clinical blood transfusion, only red blood cell ABO and RhD blood group antigens are matched for routine red blood cell, platelet and plasma transfusion, and no matching is performed for HPA, HLA and CD36. Although serological methods are simple, easy to operate and inexpensive, they cannot avoid the input and sensitization of foreign antigens. For patients who have a history of sensitization (history of blood transfusion, pregnancy, organ transplantation, etc.), need repeated blood transfusion, platelet transfusion is ineffective, have autoimmune diseases, cancer, etc., this conventional blood matching strategy can cause very serious blood transfusion complications, which is not only not conducive to the treatment of the primary disease, but also may endanger life.

[0004] In existing technologies, CN113215237A discloses a simultaneous detection kit and method for the HPA and HLA antigen systems. This method uses probe capture to perform genotyping detection of HPA and HLA-A, -B, and -C. To maximize detection coverage, this method designs and continuously optimizes a large number of probes, ultimately selecting as many as 498 probes. Furthermore, this method requires mechanical fragmentation and recapture of the gDNA extracted from each sample, resulting in a complex and costly process unsuitable for genotyping detection of large numbers of samples. Moreover, this patent only detects HPA and HLA-I class genes. CN114410758A discloses a highly efficient and specific method and kit for detecting HPA genotypes. This method uses allele-specific PCR to detect HPA genotypes, requiring multiple sets of primers for multiple-reaction tube amplification per sample, making the operation complex. It also only performs genotyping detection on HPA polymorphic sites 1–6, 15, and 21. CN111218514A discloses a method and reagent kit for determining the full-length ABO gene sequence based on next-generation sequencing technology. This method uses transposase to construct a library and perform NGS sequencing on the amplified ABO gene. The procedure is complex, and transposase fragmentation has some bias. This patent only performs ABO gene typing. In summary, there is currently no comprehensive, simple, and highly accurate genotyping method that covers all blood group antigens. Summary of the Invention

[0005] To overcome the limitations of conventional transfusion typing and the shortcomings of existing technologies, this application provides a genotyping detection method for red blood cell antigens, platelet antigens, and white blood cell antigens related to selective transfusion. This method has advantages such as comprehensive coverage, simple operation, high throughput, and high accuracy. Specifically, this invention provides the following technical solution:

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

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

[0008] (4) Enrichment of regions of interest encoding genes of multiple selective transfusion-associated antigens in one tube by using multiplex PCR amplification;

[0009] (5) Use the nucleic acid fragments enriched above to construct second- or third-generation sequencing libraries;

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

[0011] The coding genes of the selective blood 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 an 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 1 kb-35 kb, preferably 2 kb-10 kb, and further preferably 3 kb-7 kb; the number of target fragments that can be amplified by the long fragment amplification reagent ranges from 1 to 500 (the number of PCR products), preferably from 10 to 400, and further preferably from 100 to 200.

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

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

[0015] (1) An amplification system is prepared by combining the nucleic acid extracted from the sample as a template with the primers with nucleotide sequences as shown in SEQ ID NO. 1-82, and a first amplification reaction is performed to obtain a first amplification product;

[0016] (2) The first amplification product is fragmented, end-repaired and A-tailed;

[0017] (3) The product of step (2) is purified after linker ligation;

[0018] (4) The purified product is sequenced to obtain the genotyping results of the related blood cell antigens.

[0019] The method has good amplification efficiency and specificity, and there is no mutual interference between the primers when multiplex primer amplification is performed. By adjusting the ratio of the amplification primers, the concerned regions of each gene can be effectively covered by multiplex, so as to ensure the accuracy of genotyping.

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

[0021] 1. The genotyping method disclosed by the present application is an integrated typing method for multiple red blood cell, platelet and white blood cell antigen encoding genes related to blood transfusion based on DNA sequences, which can reduce the incidence of alloantigen sensitization caused by alloimmunization and promote precision blood transfusion; support blood transfusion matching for complex clinical cases: suitable for complex cases that require highly matched blood products, such as fetal neonatal immune thrombocytopenia (FNAIT) and immune thrombocytopenia patients in high-risk pregnancy;

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

[0023] 3. The method of the present application can also take into account the advantages of second and third generation sequencing: after the platform is built, it can not only perform second generation sequencing library construction, but also perform third generation sequencing library construction, providing an excellent re-creation and reproduction platform for future haplotype confirmation of multiple site variations in blood type genes, which has important significance for future precision blood transfusion. BRIEF DESCRIPTION OF DRAWINGS

[0024] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, together with the embodiments of the present application, to explain the present application, and do not constitute a limitation on the present application. In the drawings:

[0025] Figure 1 is a sequencing result graph of Example 3 of the present application. DETAILED DESCRIPTION

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

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

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

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

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

[0031] Example 1 determines the important blood type antigens related to selective blood transfusion and the screening of primers

[0032] Based on the limitations of the conventional blood transfusion matching strategy and the literature research at home and abroad, the important blood type antigens related to selective blood transfusion are determined, and the encoding genes and hotspots of the antigens involved are determined.

[0033] Through the previous retrospective study, the important blood cell antigen genes (18 genes) were determined as: 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 concerned regions, including a total of 5 parts of primer sets:

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

[0035] 1) Exon 1, exon 2, exon 3 of GP9 gene on chromosome 3 of human genome;

[0036] 2) Exon 13, exon 14, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 28, exon 29 of ITGA2 gene on chromosome 5 of human genome;

[0037] 3) Exon 16, exon 17, exon 18, exon 19, exon 20 of CD109 gene on chromosome 6 of human genome;

[0038] 4) Exon 1, exon 2 of GP1BA gene on chromosome 17 of human genome;

[0039] 5) Exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27 of ITGA2B gene on chromosome 17 of human genome;

[0040] 6) Exon 2, exon 3, exon 4, exon 5, exon 6, exon 9, exon 10, exon 11, exon 12 of ITGB3 gene on chromosome 17 of human genome;

[0041] 7) Exon 1, exon 2 of GP1BB gene on chromosome 22 of human genome.

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

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

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

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

[0046] 4) exon 6, exon 7, exon 8 of CD36 gene on chromosome 7 of the human genome;

[0047] 5) exon 9, exon 10, exon 11, exon 12, exon 13, exon 14 of CD36 gene on chromosome 7 of the human genome;

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

[0049] (iii) ABOFUT primer set amplification regions shown to include:

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

[0051] 2) exon 2, exon 3, exon 4, exon 5, exon 6 of ABO gene on chromosome 9 of the human genome;

[0052] 3) exon 7 of ABO gene on chromosome 9 of the human genome;

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

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

[0055] (iv) Rh primer set amplification regions shown to include:

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

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

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

[0059] 4) exon 2 of RHD gene and RHCE gene on chromosome 1 of the human genome;

[0060] 5) exon 3 of RHD gene and RHCE gene on chromosome 1 of the human genome;

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

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

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

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

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

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

[0067] (Five) HLA primer sets shown, including:

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

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

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

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

[0072] 5) exon1 of HLA-DRB1 gene on chromosome 6 of human genome;

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

[0074] Primer screening is the key to ensure efficient amplification, especially when the number of amplified fragments is large, scientific screening process is essential, in the optimization of primer pairs, need to consider from the primer sequence, PCR reaction system and other aspects, for this we designed and screened the primer for the above gene sites as follows:

[0075] 1. Screening of primer design

[0076] 1) Design candidate primers using dedicated primer design software (e.g. NCBIPrimer-BLAST).

[0077] 2) Exclude non-specific amplification using BLAST alignment. Optimize the uniqueness of primer target sequence for complex genomic regions.

[0078] 2. First round of screening: amplification performance of single primer pair

[0079] 1) Experimental validation: test each primer pair individually, perform PCR experiment using samples (blood donor samples with informed consent); optimize amplification conditions (e.g. annealing temperature, cycle number) to evaluate amplification efficiency and specificity.

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

[0081] After first round of screening, about 75% primer pairs pass the test and enter next round of screening.

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

[0083] 1) Performance evaluation of primer mix: mix primer pairs that pass the first round of screening, group according to fragment size; detect whether there is primer competition or interference in PCR.

[0084] 2) Screening criteria: all target product bands are clear and uniform in intensity in electrophoresis results; no primer dimer band; verify product specificity by melting curve (e.g. qPCR).

[0085] After second round of screening, about 80% primer pairs with good performance are retained and enter next round of screening.

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

[0087] 1) Fragment uniformity test: amplify different templates (e.g. mixed samples or standard samples) to 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: measure the amount of amplified product by fluorescent quantitative PCR, calculate ΔCt value to determine whether the relative efficiency of fragment amplification is consistent. Use high-throughput sequencing to verify the final product and confirm that all target regions are uniformly covered and there is no significant amplification bias.

[0089] At the third round of screening, about 90% primers can maintain good amplification effect and enter the final optimization.

[0090] 5. Final optimization: dynamic adjustment of primer sets

[0091] 1) Primer concentration optimization: adjust the concentration of each primer set according to the amplification efficiency of the fragments (e.g. reduce the concentration of highly efficient primers appropriately).

[0092] 2) Multiple rounds of dynamic optimization: combine all primers and perform a whole set of multiplex PCR, and observe the consistency of the products (ensure the consistency of the amplification products under different instruments, different experimental batches and different operators. Each product can obtain similar brightness and clear bands in electrophoresis detection experiment, and the sequencing results are consistent). According to the results, fine-tune or redesign the primers that perform poorly.

[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 experiments, the final primer set shows high stability, the amplification product band intensity is uniform, and the sequence consistency is shown in high-throughput sequencing experiment, which meets the requirements of high-quality multiplex PCR experiment).

[0094] 6. High-throughput sequencing verification

[0095] 1) Final verification of primer set: use next-generation sequencing (NGS) to evaluate the coverage, specificity and uniformity of the amplified fragments. Check for primer off-target amplification or insufficient coverage.

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

[0097] Through the above scientific and rigorous screening process, the final obtained primer set meets: all target fragments are specifically and uniformly amplified; the primer set has good compatibility, high efficiency and accurate amplification performance, and is suitable for high-throughput and standardized operation; has high stability under different experimental conditions, and can be used for analysis of complex samples and large-scale experiments.

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

[0099] Table 1: Information of the amplification primer set

[0100]

[0101]

[0102]

[0103] Case 2: Constructing NGS sequencing library using the present application

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

[0105] 1. Long fragment DNA amplification

[0106] Long fragment DNA amplification system:

[0107]

[0108] Amplification procedure:

[0109]

[0110]

[0111] After amplification, 5 μL was taken for quality control by 1% agarose gel electrophoresis. The amplification product was between 4 kb and 8 kb, indicating that the quality control was qualified. The quality control results are shown in Figure 1 After the quality control was qualified, 4 μL was taken for subsequent fragmentation.

[0112] 2. Fragmentation and end repair and A tailing (TIANSeq Direct Fast Library Kit, Tiangen, NG101)

[0113] Experimental system:

[0114]

[0115] Reaction procedure:

[0116]

[0117] 3. Adapter ligation

[0118] Adapter ligation system:

[0119]

[0120]

[0121] Adapter ligation procedure:

[0122]

[0123] 4. Purification

[0124] Purification was performed using 0.8 x 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 with 0.8x XPbeads, and the purified product was eluted with 26 μL DB and then quantified using qubit.

[0132] 7, sequencing

[0133] Double-end PE150 sequencing was performed using an illumina sequencer, and sequencing result analysis was performed. The sequencing depth results of the variant sites in the target amplified fragment of the test sample are shown in Table 2:

[0134] Table 2 Sequencing depth of target gene amplified fragment of test sample

[0135]

[0136]

[0137]

[0138] Blood group genotyping results of the test sample (Table 3):

[0139] Table 3 Blood group genotyping results of the test sample antigen

[0140]

[0141]

[0142] As can be seen from the embodiment, the method of the present application can complete the construction of a second-generation sequencing library, and the sequencing results cover the concerned regions of 18 genes of human selective blood 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 qubit quantification was performed, and long fragment DNA amplification was performed

[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 purification. After purification, elute with 20 μL of DB. Take 5 μL of the eluted product for quality control by 1% agarose gel electrophoresis. After quality control, take 500 ng for subsequent library construction.

[0151] 2. End repair and A tailing

[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 linker ligation

[0157] The ligation system is as follows:

[0158]

[0159]

[0160] The ligation procedure is as follows:

[0161]

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

[0163] 4. Purification

[0164] Purification is performed with 0.4 x XPbeads, and the purified product is eluted with 35 μL of DB.

[0165] 5. Adaptor linker ligation

[0166] The ligation system is as follows:

[0167]

[0168] The ligation procedure is as follows:

[0169]

[0170] 6. Purification

[0171] Purification was performed with 0.4x XP beads, washes were performed with Long Fragment Buffer (LFB) from the Nanopore kit, and the purified product was eluted with 15 μΐ, of DB and quantified with qubit.

[0172] 7, sequencing

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

[0174] Table 4 Comparison of the results of the blood group genotyping of test sample 3 obtained with the two sequencing methods

[0175]

[0176]

[0177] The sequencing results of Example 3 show that the library construction and sequencing of the third generation sequencing can be completed using the method of the present application. The results of the third generation Nanopore sequencing of test sample 3 are consistent with the results of the second generation sequencing ( Figure 1 ). The second generation sequencing (NGS) has difficulty in haplotype assembly due to short read length, which can produce ambiguous results, especially in regions of high polymorphism and complex structural variation, while the third generation sequencing (such as Nanopore) has the advantage of long read length, which can directly cover the entire gene region and accurately analyze whether the variation is located on the same chromosome, providing clear information for haplotype assembly. Therefore, the present application can not only accurately identify the variation site on the basis of third generation sequencing, but also identify the haplotype of the variation site, and accurately determine the typing of complex genes.

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

Claims

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

2. The use of the primer set as described in claim 1 in the preparation of an integrated genotyping kit, characterized in that, The integrated genotyping kit is for typing multiple blood cell antigens associated with selective transfusion. The method of using the kit includes: (1) Enrichment of regions of interest encoding genes of multiple selective transfusion-associated antigens in one tube by multiplex PCR amplification; (2) Using the nucleic acid fragments enriched above, construct second- or third-generation sequencing libraries; (3) The genes of relevant blood cell antigens are genotyped.

3. The use as described in claim 2, characterized in that, The genes encoding the selective transfusion-associated multiple blood cell antigens include ITGB3, GP1BA, GP1BB, ITGA2B, ITGA2, CD109, GP9, HLA-A, -B, -C, -DRB1, -DQB1, ABO, FUT1, FUT2, RhD, RhCE, and CD36.

4. The use as described in claim 2, characterized in that, The primer mixture used in step (1) for multiplex PCR amplification is a long fragment amplification reagent with an amplification length range of 1kb-35kb; the long fragment amplification reagent can amplify the target fragment with a multiplicity range of 1-500.

5. The use as described in claim 4, characterized in that, The amplification length range is 2kb-10kb, and the multiplicity of the target fragment that the long fragment amplification reagent can amplify ranges from 10 to 400.

6. The use as described in claim 5, characterized in that, The amplification length range is 3kb-7kb, and the multiplicity of the target fragment that the long fragment amplification reagent can amplify ranges from 100 to 200.

7. The use as described in any one of claims 2-6, characterized in that, The primers are as shown in SEQ ID NO.1-82.

8. The use as described in any one of claims 2-6, characterized in that, The specific steps for using the kit are as follows: (1) Using the nucleic acid extracted from the sample as a template, an amplification system was prepared with the primer sets with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.82, and the first amplification reaction was carried out to obtain the first amplification product; (2) Fragment the first amplification product and repair and fill in the ends by adding an A tail; (3) The product from step (2) is purified after ligation with a linker; (4) Sequencing of the purified product to obtain the genotyping results of the relevant blood cell antigens.

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