Gene typing analysis method, probe set and kit

Abstract

The invention discloses a genotyping analysis method, a probe set and a kit. The method comprises the following steps: designing and synthesizing a probe sequence set for capturing a coding region of 75 genes, a 5 '-end non-coding region and a cleavage site, and carrying out genotyping and variation site identification on an erythrocyte system by using a BGAseq blood group system analysis platform. According to the present invention, three different antigen types (HEA, HPA and HNA) related genes can be identified, wherein 53 red blood cell blood group system related genes, 6 platelet related genes, 5 neutrophil related genes and 11 blood group related genes are published by the International Transfusion Association; the kit comprises a probe set of 75 genes, can capture and enrich corresponding target gene sequences in human genomes, reduces interference of non-target sequences, achieves capture and sequencing of 75 blood type related genes in the same reaction system, and achieves accurate blood type typing.

Description

Technical Field

[0001] The present invention relates to the technical field of genotyping technology detection, and specifically relates to a genotyping analysis method, a probe set and a kit. Background Art

[0002] Human red blood cells (RBC), platelets (PLT) and neutrophil antigens are of great significance in clinical blood transfusion, forensic medicine identification and obstetric medicine, etc. When mismatched red blood cells or platelet transplant cells are transfused, patients with alloantibodies against "non-self" antigens are at risk of hemolytic transfusion reactions. Pregnant women with red blood cell, platelet or neutrophil alloantibodies have a risk of having a baby with hemolytic disease of the newborn, neonatal alloimmune thrombocytopenia or neonatal alloimmune neutropenia. Therefore, accurate blood group screening and identification technology can ensure blood transfusion safety and protect the life and health of blood transfusion recipients.

[0003] In blood group antigen genotyping and identification, serological techniques are widely used clinically. However, this method has some limitations. For example, currently commercially available monoclonal serum antibodies only cover about 10% of blood group antigens, making it difficult to identify complex blood groups and not suitable for high-throughput screening of large populations. Therefore, the importance of high-throughput molecular diagnostic techniques in blood group typing has become increasingly obvious. Currently, blood group typing molecular diagnostic techniques mainly include methods such as PCR-SSP and PCR-SSOP. However, the target genes and targets of the blood group genes detected by these methods are relatively single and cannot cover all the target gene sequences and targets of blood group typing at the same time.

[0004] Dozens of blood group system genes are involved in blood group typing, and the corresponding gene mutation types and phenotypes are intricate. Current methods only focus on mutations in the coding region. However, the regulatory expression in the 5′ non-coding region and the influence of splicing sites on mRNA transcription will both affect the final blood group antigen phenotype. Therefore, from the perspective of ensuring blood transfusion safety, there is an urgent need for a high-throughput blood group typing diagnostic technology that can simultaneously detect all blood group-related genes and enable rapid detection of large populations, so as to help achieve high-throughput and high-precision blood group typing and ensure blood safety.

[0005] Targeted next-generation sequencing (tNGS) can enrich specific target regions in the genome for accurate analysis through probe hybridization capture technology or multiplex PCR technology, and has the characteristics of high sensitivity, high detection throughput, and low cost. Among them, the hybridization capture sequencing method based on probe capture can simultaneously capture a large number of target genes in a sample by designing capture probes for specific target genes, and has high coverage and sensitivity. It is suitable for the detection of single nucleotide variations, translocations, structural variations, insertions and deletions, and copy number variations, and is suitable for high-throughput blood group genotyping detection of large populations.

[0006] For this reason, a genotyping analysis method is proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a genotyping analysis method to solve the problems raised in the above background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solution: A genotyping analysis method, comprising the following steps:

[0009] S1. Design and synthesize a probe sequence set that captures the coding regions, 5'-untranslated regions, and splice sites of 75 genes;

[0010] S2. Extract and prepare genomic gDNA from blood;

[0011] S3. Fragment the genomic DNA using the enzymatic digestion method, and at the same time perform end repair and A-tailing of the DNA fragments;

[0012] S4. Use the DNA fragmentized product after end repair in S3 for adapter ligation, and purify the ligation product using magnetic beads;

[0013] S5. Perform PCR amplification enrichment on the purified product in S4, and at the same time attach a sample tag sequence adapter to each fragment. After the reaction, perform magnetic bead purification;

[0014] S6. Accurately quantify the product in S5 using a Qubit fluorescence quantifier;

[0015] S7. Perform liquid-phase hybridization on the purified product in S4 using the probe designed and synthesized in step S1;

[0016] S8. Capture the product in S6 using streptavidin magnetic beads, and remove non-specific fragments and impurities through heat elution and room temperature elution steps;

[0017] S9. Perform PCR enrichment on the product in S7, and perform magnetic bead purification on the PCR product to obtain a capture library.

[0018] S10. Accurately quantify the captured library using a Qubit fluorometer.

[0019] S11. Use the MGIDNBSEQ one-step DNB preparation kit and the DNBSEQ high-throughput sequencing kit to prepare DNBs from the captured library and perform on-machine sequencing on the MGI sequencing platform.

[0020] S12. Perform bioinformatics analysis on the off-machine data obtained in S11, and use the BGAseq blood group system analysis platform to genotype the erythrocyte system genes and identify variant sites.

[0021] Preferably, the 75 gene sets included in S1 are as follows: 53 genes come from 47 red blood cell blood group systems: the ABO gene of the 001 ABO blood group system, the GYPA, GYPB, and GYPE genes of the 002 MNS blood group system, the A4GALT gene of the 003 P1PK system, the RHD and RHCE genes of the 004 Rh blood group system, the BCAM gene of the 005 Lutheran system, the KEL gene of the 006 Kell system, the FUT3 of the 007 Lewis system, the ACKR1 gene of the 008 FY system, the SLC14A1 gene of the 009 JK system, the SLC4A1 gene of the 010 DI system, the ACHE gene of the 011 YT system, the XG and CD99 genes of the 012 Xg system, the ERMAP gene of the 013 Scianna system, the ART4 gene of the 014 Dombrock system, the CO gene of the 015 CO system, the ICAM4 gene of the 016 Landsteiner-Wiener system, the C4A and C4B genes of the 017 Chido / Rodgers system, the FUT1 and FUT2 genes of the 018 H system, the XK gene of the 019 Kx system, the GYPC gene of the 020 Gerbich system, the CD55 gene of the 021 Cromer system, the CR1 gene of the 022 Knops system, the CD44 gene of the 023 Indian system, the BSG gene of the 024 OK system, the CD151 gene of the 025 Raph system, the SEMA7A gene of the 026 John Milton Hagen system, the GCNT2 gene of the 027 I system, the B3GALNT1 gene of the 028 Globoside system, the AQP3 gene of the 029 Gill system, the RHAG gene of the 030 Rh-associated glycoprotein system, the GBGT1 gene of the 031 FORS system, the ABCG2 gene of the 032 JR system, the ABCB6 gene of the 033 LAN system, the SMIM1 gene of the 034 Vel system, the CS59 gene of the 035 CD59 system, the SLC29A1 gene of the 036 Augustine system, the PRNP gene of the 037 KANNO system, the B4GALNT2 gene of the 038 SID system, the SLC44A2 gene of the 039 CTL2 system, the ABCC4 gene of the 040 PEL system, the EMP3 gene of the 041 MEM system, the PIGG gene of the 042 EMM system, the ABCC1 gene of the 043 ABCC1 system, the PIEZO1 gene of the 044 Er system, the CD36 gene of the 045 CD36 system, the ATP11C gene of the 046 ATP11C system, and the MAL gene of the 047 MAL system;

[0022] 6 blood type-related genes from 35 human platelets: ITGB3, GP1BA, ITGA2B, ITGA2, GP1BB, and CD109;

[0023] 5 genes from 5 neutrophil systems: FCGR3B, CD177, SLC44A2, ITGAM, and ITGAL;

[0024] 11 other blood type-related genes: KLF1, GATA1, SMIM1 eQTL, TMEM50A, SLC35C1, PIGA, C1GALT1, C1GALT1C1, GP9, CD36, ITGB2.

[0025] Preferably, all hybridization capture probes in S1 are conjugated with streptavidin at the 5′ end and uniformly mixed into one tube for use.

[0026] Preferably, the blood genomic preparation kit in S2 is the MGIEasy Blood Genomic DNA Extraction Kit.

[0027] Preferably, the WGS library preparation kit used for hybridization capture in S3 to S5 is the HieffNGS OnePot Pro DNA Library Prep Kit from Yeasen Biotech.

[0028] Preferably, in steps S7 to S9, the probe hybridization capture reagent is the DNA hybridization capture kit from Nano & Gene, and the magnetic bead reagent used in the magnetic bead purification steps in S4, S5, and S9 is the MGI DNA Clean Beads magnetic beads.

[0029] Preferably, the library concentration determination reagent in S6 and S10 is the 1× dsDNA HS Assay Kit double-stranded DNA quantification reagent from Yeasen Biotech. In S11, the MGI sequencing platform and the high-throughput sequencing kit are the DNBSEQ-E25RS and DNBSEQ-E25R high-throughput sequencing reagent sets.

[0030] Preferably, a kit includes a detection kit for synchronous capture of the coding regions, 5′ untranslated regions, and splice site sequences of 75 blood type-related genes. The 63 blood type-related gene synchronous detection kit includes a probe set with nucleotide sequences shown in SEQ ID NO.1 to ID NO.2023 and streptavidin-labeled at the 5′ end, as well as reagents for library construction and sequencing on the machine.

[0031] Preferably, a probe set includes a probe set that simultaneously captures the coding regions, 5'-untranslated regions, and splice site sequences of 75 genes, and the nucleotide sequences of the probe set are as shown in SEQ ID NO.1 to ID NO.2023.

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[0146] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0147] 1. The present invention can identify genes related to 3 different antigen types (HEA, HPA, and HNA), including 53 genes related to the red blood cell blood group system, 6 platelet-related genes, 5 neutrophil-related genes, and 11 genes associated with blood groups announced by the International Society of Blood Transfusion. The probe set for the above 75 genes is included in the kit of the present invention, which can achieve the capture and enrichment of the corresponding target gene sequences in the human genome, reduce the interference of non-target sequences, and realize the capture and sequencing of 75 blood group-related genes in the same reaction system, so as to achieve accurate blood group typing.

[0148] 2. In the method of the present invention, in addition to capturing the coding regions of each gene, the probes can also capture the 5′-untranslated regions and splicing sites of the genes, and can analyze the non-coding regions related to antigen expression in genomic DNA, so as to judge the influence of the non-coding regions and achieve accurate blood group antigen typing.

[0149] 3. The method of the present invention can identify the cis-trans relationship of exons 6 and 7 of the ABO gene. The main SNP sites for ABO gene typing are located in the sequences of exons 6 and 7. If probes are designed only for exons 6 and 7 respectively, it is difficult to determine the cis-trans relationship between exons 6 and 7 from the analysis results. Therefore, we use the 6th exon and the 7th exon of the ABO gene, as well as the intron between the two exons, as a target region for probe design. During bioinformatics analysis, the sequences of exons 6 and 7 are assembled and their cis-trans relationship is analyzed, so as to achieve more accurate identification of the ABO genotype.

[0150] 4. The probes involved in the method of the present invention shield the repetitive sequences with high risk for capture in the target sequence to achieve higher capture efficiency and effective data ratio. In addition, probe encryption design is carried out for the low GC (=<40% GC) regions with weak binding ability to the probes to ensure the uniformity of the sequencing results and truly reflect the situation of the target sequence.

[0151] 5. The probes designed by the method of the present invention are designed for the hg19 version reference genome. During the probe capture process, multiple base mismatches are allowed, so as to capture the relevant coding region and non-coding region sequences of different alleles, and thus unknown harmful variations in the target gene can be mined.

[0152] 6. The detection method provided by the present invention can solve the problem that the existing blood type detection means on the market have a single covered target site and it is difficult to achieve high-throughput screening. It has the characteristics of accurate detection, wide coverage and large-scale screening, and has important significance in clinical blood transfusion applications and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 It is the Agilent 2100 library fragment peak map of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0154] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0155] Please refer to Figure 1 , the present invention provides a technical solution for a genotyping analysis method:

[0156] A genotyping analysis method includes the following steps:

[0157] S1. Design and synthesize a probe sequence set that captures the coding regions, 5'-untranslated regions, and splice sites of 75 genes;

[0158] S2. Extract and prepare genomic DNA (gDNA) from blood;

[0159] S3. Fragment the genomic DNA using the enzymatic digestion method, and simultaneously perform end repair and A-tailing of the DNA fragments;

[0160] S4. Perform adapter ligation on the fragmented DNA products after end repair in S3, and purify the ligation products using magnetic beads;

[0161] S5. Perform PCR amplification and enrichment on the purified products in S4, and simultaneously attach sample tag sequence adapters to each fragment. After the reaction, perform magnetic bead purification;

[0162] S6. Accurately quantify the products in S5 using a Qubit fluorometer;

[0163] S7. Perform liquid hybridization on the purified products in S4 using the probes designed and synthesized in step S1;

[0164] S8. Capture the products in S6 using streptavidin magnetic beads, and remove non-specific fragments and impurities through heat elution and room temperature elution steps;

[0165] S9. Perform PCR enrichment on the products in S7, and purify the PCR products using magnetic beads to obtain a captured library.

[0166] S10. Accurately quantify the captured library using a Qubit fluorometer.

[0167] S11. Perform DNB preparation on the captured library using the MGIDNBSEQ one-step DNB preparation kit and the DNBSEQ high-throughput sequencing kit, and perform on-machine sequencing on the MGI sequencing platform.

[0168] S12. Perform bioinformatics analysis on the off-machine data obtained in S11, and use the BGAseq blood group system analysis platform to genotype genes in the erythrocyte system and identify variant sites.

[0169] The 75 gene sets included in S1, among which: 53 genes come from 47 red blood cell blood group systems: the ABO gene of the 001 ABO blood group system, the GYPA, GYPB, and GYPE genes of the 002 MNS blood group system, the A4GALT gene of the 003 P1PK system, the RHD and RHCE genes of the 004 Rh blood group system, the BCAM gene of the 005 Lutheran system, the KEL gene of the 006 Kell system, the FUT3 of the 007 Lewis system, the ACKR1 gene of the 008 FY system, the SLC14A1 gene of the 009 JK system, the SLC4A1 gene of the 010 DI system, the ACHE gene of the 011 YT system, the XG and CD99 genes of the 012 Xg system, the ERMAP gene of the 013 Scianna system, the ART4 gene of the 014 Dombrock system, the CO gene of the 015 CO system, the ICAM4 gene of the 016 Landsteiner-Wiener system, the C4A and C4B genes of the 017 Chido / Rodgers system, the FUT1 and FUT2 genes of the 018 H system, the XK gene of the 019 Kx system, the GYPC gene of the 020 Gerbich system, the CD55 gene of the 021 Cromer system, the CR1 gene of the 022 Knops system, the CD44 gene of the 023 Indian system, the BSG gene of the 024 OK system, the CD151 gene of the 025 Raph system, the SEMA7A gene of the 026 John Milton Hagen system, the GCNT2 gene of the 027 I system, the B3GALNT1 gene of the 028 Globoside system, the AQP3 gene of the 029 Gill system, the RHAG gene of the 030 Rh-associated glycoprotein system, the GBGT1 gene of the 031 FORS system, the ABCG2 gene of the 032 JR system, the ABCB6 gene of the 033 LAN system, the SMIM1 gene of the 034 Vel system, the CS59 gene of the 035 CD59 system, the SLC29A1 gene of the 036 Augustine system, the PRNP gene of the 037 KANNO system, the B4GALNT2 gene of the 038 SID system, the SLC44A2 gene of the 039 CTL2 system, the ABCC4 gene of the 040 PEL system, the EMP3 gene of the 041 MEM system, the PIGG gene of the 042 EMM system, the ABCC1 gene of the 043 ABCC1 system, the PIEZO1 gene of the 044 Er system, the CD36 gene of the 045 CD36 system, the ATP11C gene of the 046 ATP11C system, the MAL gene of the 047 MAL system;

[0170] 6 blood group-related genes from 35 human platelets: ITGB3, GP1BA, ITGA2B, ITGA2, GP1BB, and CD109;

[0171] 5 genes from 5 neutrophil systems: FCGR3B, CD177, SLC44A2, ITGAM, and ITGAL;

[0172] 11 other blood group-related genes: KLF1, GATA1, SMIM1 eQTL, TMEM50A, SLC35C1, PIGA, C1GALT1, C1GALT1C1, GP9, CD36, ITGB2;

[0173] All hybridization capture probes in S1 are conjugated with streptavidin at the 5′ end and uniformly mixed into one tube for use;

[0174] The blood genomic preparation kit in S2 is the MGIEasy Blood Genomic DNA Extraction Kit;

[0175] The WGS library preparation kit used for hybridization capture in S3 to S5 is the HieffNGS OnePot Pro DNA Library Prep Kit from Yeasen Biotech;

[0176] In steps S7 to S9, the probe hybridization capture reagent is the DNA hybridization capture kit from Nanoanda, and the magnetic bead reagent used in the magnetic bead purification steps in S4, S5, and S9 is the MGI DNA Clean Beads;

[0177] The library concentration determination reagent in S6 and S10 is the 1× dsDNA HS Assay Kit double-stranded DNA quantification reagent from Yeasen Biotech. In S11, the MGI sequencing platform and the high-throughput sequencing kit are the DNBSEQ-E25RS and DNBSEQ-E25R high-throughput sequencing reagent sets;

[0178] S2. Preparation of blood genomic (gDNA) samples

[0179] Aspirate 200 μL of the blood sample to be tested, extract genomic DNA according to the MGIEasy Blood Genomic DNA Extraction Kit (magnetic bead method), and measure the concentration and purity of the genomic DNA.

[0180] S3. DNA fragmentation / end repair / dA tail addition

[0181] Use the YEASEN Hieff NGS OnePot Pro DNA Library Prep Kit V3 to prepare the reaction system according to Table 1 below. Note that operations should be carried out on ice. After adding each component to the PCR tube, gently pipette or vortex at low speed.

[0182] Table 1 Reaction system for DNA fragmentation / end repair / dA tail addition

[0183] Name Volume / μL Input DNA X (100 ng) Smearase Buffer 3.0 10 Smearase Enzyme 3.0 10 <![CDATA[ddH 2 O]]> Up to 60

[0184] After the reaction system is prepared, place it in a PCR instrument pre-cooled to 4°C, start the reaction program, and set the reaction program as shown in Table 2.

[0185] Table 2 Reaction program for library enzymatic digestion and fragmentation

[0186] Temperature Time Hot lid 105 °C On 4℃ 1 min 37℃ 15 min 72℃ 20 min 4℃ Hold

[0187] S4. Adapter ligation

[0188] Prepare the adapter ligation reaction system in a PCR tube according to Table 3, gently pipette and then centrifuge briefly and place on ice.

[0189] Table 3 Adapter ligation reaction system

[0190] Name Volume / μL dA-tailed DNA (product of step 2) 60 Ligation Ready Mix 25 DNA Adapter 5

[0191] Place the PCR tube in the PCR instrument and set the PCR instrument for adapter ligation reaction according to Table 4.

[0192] Table 4 Adapter ligation reaction program

[0193] Temperature Time Hot lid Off 20℃ 15 min 4℃ Hold

[0194] S5. Magnetic bead purification of adapter ligation products

[0195] The purification of the ligation product was carried out using the MGIEasy DNA Purification Magnetic Bead Kit. The DNA Clean Beads were taken out in advance and placed at room temperature for 30 min. Mix the DNA Clean Beads well, pipette 72 μL of DNA Clean Beads into the ligation product for mixing, and incubate at room temperature for 5 min. After briefly centrifuging the PCR tube, place it on the magnetic rack and let it stand for 2 - 5 min until the liquid becomes clear, then aspirate and discard the supernatant. Keep the PCR tube / plate fixed on the magnetic rack, add 200 μL of 80% ethanol to wash the magnetic beads, let it stand for 30 s and then aspirate and discard the supernatant. Repeat the ethanol washing step once. Try to suck out as much residual liquid in the tube as possible. Keep the PCR tube fixed on the magnetic rack, open the tube cap and dry it at room temperature until there is no reflection and no cracking on the surface of the magnetic beads. Remove the PCR tube from the magnetic rack, add 21 μL of TE Buffer for DNA elution, gently pipette at least 10 times until all the magnetic beads are suspended. Incubate at room temperature for 5 min, remove the PCR tube from the magnetic rack, add 21 μL of TE Buffer for DNA elution, quickly pipette at least 10 times until completely mixed, and avoid generating bubbles during the process. Place the PCR tube on the magnetic rack, let it stand for 2 - 5 min until the liquid becomes clear, and transfer 20 μL of the supernatant to a new 0.2 mL PCR tube.

[0196] S6, DNA Library Amplification

[0197] Prepare the reaction system in a PCR tube according to Table 5, gently pipette to mix well, and then briefly centrifuge and place on ice.

[0198] Table 5 DNA Library Amplification Reaction System

[0199] Name Volume / μL Adapter Ligated DNA (product of step 4) 20 2X Ultima HF Amplification Mix 25 Primer Mix (for MGI) 5

[0200] Place the PCR tube in the PCR instrument, set the reaction program according to Table 6, and perform PCR amplification.

[0201] Table 6 DNA Library Amplification Reaction Program

[0202]

[0203] S7, Purification of DNA Library Amplification Product

[0204] The purification of the DNA library amplification product was carried out using the MGIEasy DNA Purification Magnetic Bead Kit. The DNA CleanBeads were taken out in advance and placed at room temperature for 30 min. The DNA CleanBeads were mixed well, and 50 μL of DNA CleanBeads was pipetted into the ligation product for mixing, followed by incubation at room temperature for 5 min. After briefly centrifuging the PCR tube, it was placed on a magnetic rack and left to stand for 2 - 5 min until the liquid became clear. The supernatant was aspirated and discarded. Keeping the PCR tube / plate fixed on the magnetic rack, 200 μL of 80% ethanol was added to wash the magnetic beads. After standing for 30 s, the supernatant was aspirated and discarded, and the ethanol washing step was repeated once. Try to dry the residual liquid in the tube as much as possible. Keeping the PCR tube fixed on the magnetic rack, the tube lid was opened and dried at room temperature until there was no reflection and no cracking on the surface of the magnetic beads. The PCR tube was removed from the magnetic rack, 21 μL of TE Buffer was added for DNA elution, and the pipette was used to gently pipette at least 10 times until all the magnetic beads were suspended. Incubate at room temperature for 5 min. The PCR tube was removed from the magnetic rack, 21 μL of TE Buffer was added for DNA elution, and the pipette was used to quickly pipette at least 10 times until thoroughly mixed, and bubbles should be avoided during the process. The PCR tube was placed on the magnetic rack and left to stand for 2 - 5 min until the liquid became clear, and 20 μL of the supernatant was transferred to a new 0.2 mL PCR tube.

[0205] S8, Quality Control of DNA Library

[0206] The dsDNA HS Assay Kit for Qubit double-stranded DNA fluorescence quantitative kit was used to quantify the purified product of the DNA library according to the operation instructions of the quantitative kit. The fragment distribution of the product after PCR purification was detected by Bioanalyzer 2100.

[0207] S9, Vacuum Concentration - Hybridization of Library

[0208] The vacuum concentration instrument was preheated to 60 °C in advance. The vacuum concentration reaction system was prepared in a low-adsorption 1.5 mL EP tube according to Table 7. After vortex mixing and brief centrifugation, the EP tube was placed in the vacuum concentrator and dried for 10 - 30 min until all the liquid evaporated.

[0209] Table 7 Vacuum Concentration Reaction System

[0210] Name Volume / μL WGS Library 500 ng Human Cot DNA 5 BLG Universal Blocker - MGI 2

[0211] ES Hyb#1, Hyb#2 and NEXome Core Panel were taken out in advance and thawed. The hybridization reaction solution was prepared according to Table 8. After vortex mixing and brief centrifugation, 17 μL of the hybridization reaction solution was added to the bottom of the EP tube dried by vacuum concentration. After gently pipetting and mixing 10 - 20 times, it was briefly centrifuged and incubated at 25 °C for 10 min.

[0212] Table 8 Hybridization Reaction Solution

[0213] Name Volume / μL ESHyb #1 8.5 Hyb #2 2.7 Nuclease Free Water 1.8 NEXome Core Panel 4

[0214] After the incubation is completed, transfer the hybridization reaction solution in the EP tube to a new 0.2 mL PCR tube, centrifuge briefly, and place it in a PCR instrument, and perform the hybridization reaction according to Table 9.

[0215] Table 9 Hybridization Reaction Program

[0216] Temperature Time Number of cycles Hot lid 100 °C On / 65℃ 4h 1 65℃ Hold /

[0217] S10. Washing of Streptavidin Magnetic Beads

[0218] Take out the ES Wash Buffer in advance and let it melt naturally. Aliquot two portions of 160 μL ES Wash Buffer and incubate in a PCR instrument at 65 °C until use. Place the Steptavidin Beads at room temperature and equilibrate for 30 min. Prepare the magnetic bead suspension according to Table 10.

[0219] Table 10 Magnetic Bead Suspension

[0220] Name Volume / μL ESHyb #1 8.5 Hyb #2 2.7 Nuclease Free Water 5.8

[0221] Vortex the Steptavidin Beads for 15 s, then pipette 50 μL of the magnetic beads into a 0.2 mL PCR tube. Then add 100 μL of ES Wash Buffer (at room temperature), gently pipette up and down 10 times, centrifuge briefly, and place on a magnetic stand for 1 - 2 min until the liquid is clear. Aspirate and discard the supernatant, and repeat the washing step once. Then add 17 μL of the magnetic bead suspension to the PCR tube, gently pipette up and down to mix, and transfer all the solution to a new 0.2 mL PCR tube, and incubate in a PCR instrument at 65 °C for more than 5 min.

[0222] S11. Streptavidin Capture

[0223] Add all of the magnetic bead suspension incubated at 65 °C to the hybridization reaction solution, gently pipette up and down to mix, then incubate at 65 °C for 45 min, and vortex at low speed for 5 s every 12 min to ensure that the magnetic beads are completely resuspended.

[0224] S12. Thermal Elution of Captured Magnetic Beads

[0225] After the incubation is completed, open the PCR tube on a PCR instrument, add 150 μL of 65°C ES Wash Buffer, vortex mix at low speed, place it on a magnetic stand, quickly aspirate and discard the supernatant after the liquid becomes clear, place the PCR tube back into the PCR instrument, and add another 150 μL of 65°C ES Wash Buffer, then vortex mix at low speed. Then transfer all the solution to a new low-binding 1.5 mL EP tube, and incubate it at 65°C and 1200 rpm for 5 min in a thermostatic shaker.

[0226] S13. Room temperature elution of capture beads

[0227] Place the 1.5 mL EP tube in a magnetic stand for 30 s. After the liquid becomes clear, aspirate and discard the supernatant. Add 150 μL of room temperature ES Wash Buffer, gently pipette 10 - 15 times, and transfer all the resuspended liquid to a new low-binding 1.5 mL EP tube. Incubate at room temperature for 2 min, during which vortex at low speed for 30 s and then let it stand for 30 s, alternating to ensure complete mixing. After the incubation is completed, place the 1.5 mL EP tube in a magnetic stand for 30 s. After the liquid becomes clear, aspirate and discard the supernatant. Add 150 μL of room temperature ES Wash Buffer, gently pipette 10 - 15 times. Incubate at room temperature for 2 min, during which vortex at low speed for 30 s and then let it stand for 30 s, alternating to ensure complete mixing. Place the 1.5 mL EP tube in a magnetic stand for 30 s. After the liquid becomes clear, aspirate and discard the supernatant, and then perform a short centrifugation and thoroughly aspirate the residual liquid at the bottom of the tube. Remove the PCR tube from the magnetic stand, add 22.5 μL of Nuclease Free Water, and gently pipette to mix.

[0228] S14. PCR amplification of capture library

[0229] Prepare the PCR amplification reaction system in Table 11 on ice, vortex mix and then perform a short centrifugation, and place it on ice.

[0230] Table 11 PCR amplification reaction system

[0231]

[0232] Place the PCR tube in a PCR instrument and perform amplification according to the PCR program set in Table 12.

[0233] Table 12 PCR amplification reaction program

[0234]

[0235] S15. Quality inspection of capture library

[0236] Use the dsDNA HS Assay Kit for Qubit to quantify the purified product of the capture library according to the operation instructions of the quantification kit. Detect the fragment distribution of the purified product of the capture library by Bioanalyzer 2100.

[0237] S16, Make DNB

[0238] Prepare DNB reaction system 1 in a 0.2 mL PCR tube according to Table 13. After vortexing and mixing, centrifuge instantaneously and place on ice.

[0239] Table 13 DNB reaction system 1

[0240] Name Volume / μL Captured Library X (30 ng) Low TE buffer 20-X Make DNB Buffer (OS, DB) 20

[0241] Place the PCR tube in the PCR instrument and set the reaction program according to Table 14 for reaction.

[0242] Table 14 Reaction program of DNB reaction system 1

[0243] Temperature Time Number of cycles Hot lid 105 °C On / 95℃ 3 min 1 40℃ 3 min 1 4℃ Hold /

[0244] Then, according to Table 15, prepare DNB reaction system 2. After vortexing and mixing, centrifuge instantaneously and place on ice.

[0245] Table 15 DNB reaction system 2

[0246] Name Volume / μL DNB Polymerase Mix I 40 DNB Polymerase Mix II 4

[0247] Then add 44 μL of the DNB reaction system 2 solution to the PCR tube. After vortexing and mixing, centrifuge instantaneously. Place the PCR tube in the PCR instrument and set the reaction program according to Table 16 for reaction. After the reaction is completed, add 20 μL of DNB termination buffer and gently pipette and mix with a wide-mouth pipette tip.

[0248] Table 16 Reaction program of DNB reaction system 2

[0249] Temperature Time Number of cycles Hot lid 35 On / 30℃ 25 min 1 4℃ Hold /

[0250] S17, DNB quality inspection

[0251] Use the ssDNA HS Assay Kit for Qubit to quantify the DNB concentration according to the operation instructions of the quantification kit.

[0252] S18, Sequencing on the machine

[0253] After quantifying the DNB, use the DNBSEQ-E25RS and PE150 sequencing kits to perform sequencing analysis on the capture library.

[0254] As an embodiment of the present invention

[0255] A kit, comprising a detection kit for synchronous capture of coding regions, 5'-untranslated regions and splice site sequences of 75 blood group-related genes. The synchronous detection kit for 63 blood group-related genes includes nucleotide sequences shown in SEQ ID NO.1 to ID NO.2023, and a probe set labeled with streptavidin coupled to the 5' end, as well as reagents for library construction and sequencing on a machine.

[0256] As an embodiment of the present invention

[0257] A probe set, comprising a probe set for synchronous capture of coding regions, 5'-untranslated regions and splice site sequences of 75 genes. The nucleotide sequences of the probe set are as shown in SEQ ID NO.1 to ID NO.2023.

[0258] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A genotyping analysis method, characterized in that: The following steps are involved: S1. Design and synthesize a probe sequence set that captures the coding region, 5′ non-coding region, and splicing site of 75 genes; S2, extracting and preparing blood genomic gDNA; S3, fragment the genomic DNA using enzyme shearing method, and simultaneously perform DNA fragment end repair and A-tailing; S4, using the end-repaired DNA fragmentation products of S3 to perform adapter ligation, and purifying the ligation products by magnetic beads; S5, PCR amplification and enrichment of the purified product of S4, and at the same time, each fragment is provided with a sample tag sequence adapter, and magnetic bead purification is performed after the reaction is completed; S6. Use Qubit fluorescence quantification instrument to accurately quantify the product of S5; S7, using the probe designed and synthesized in step S1 to perform liquid phase hybridization on the purified product in step S4; S8, using streptavidin magnetic beads to capture the S6 product, and removing non-specific fragments and impurities through heat elution and room temperature elution steps; S9, performing PCR enrichment on the product of step S7, and performing magnetic bead purification on the PCR product to obtain a capture library; S10, accurately quantify the captured library using the Qubit fluorescence quantifier; S11, using MGIDNBSEQ one-step DNB preparation kit and DNBSEQ high-throughput sequencing kit to prepare DNBs from the capture library, and perform sequencing on the MGI sequencing platform; S12. Perform bioinformatics analysis on the offline data obtained in S11, and use the BGAseq blood type system analysis platform to perform red blood cell system gene typing and variant site identification.

2. A genotyping analysis method according to claim 1, characterized in that: The 75 gene sets included in the S1 include: 53 genes from 47 red blood cell blood group systems: 001 ABO gene of the ABO blood group system, 002 GYPA, GYPB and GYPE genes of the MNS blood group system, 003 A4GALT gene of the P1PK system, 004 RHD and RHCE genes of the Rh blood group system, 005 BCAM gene of the Lutheran system, 006 KEL gene of the Kell system, 007 FUT3 of the Lewis system, 008 ACKR1 gene of the FY system, 009 SLC14A1 gene of the JK system, 010 SLC4A1 gene of the DI system, 011 ACHE of the YT system gene, 012Xg system XG and CD99 genes, 013Scianna system ERMAP gene, 014Dombrock system ART4 gene, 015CO system CO gene, 016Landsteiner-Wiener system ICAM4 gene, 017Chido / Rodgers system C4A and C4B genes, 018H system FUT1 and FUT2 genes, 019Kx system XK gene, 020Gerbich system GYPC gene, 021Cromer system CD55 gene, 022Knops system CR1 gene, 023Indian system 024 CD44 gene of OK system, 025 BSG gene of Raph system, 026 SEMA7A gene of JohnMiltonHagen system, 027 GCNT2 gene of I system, 028 B3GALNT1 gene of Globoside system, 029 AQP3 gene of Gill system, 030 RHAG gene of Rh-associatedglycoprotein system, 031 GBGT1 gene of FORS system, 032 ABCG2 gene of JR system, 033 ABCB6 gene of LAN system, 034 SMIM1 gene of Vel system, 035 5CS59 gene of CD59 system, 036SLC29A1 gene of Augustine system, 037PRNP gene of KANNO system, 038B4GALNT2 gene of SID system, 039SLC44A2 gene of CTL2 system, 040ABCC4 gene of PEL system, 041EMP3 gene of MEM system, 042PIGG gene of EMM system, 043ABCC1 gene of ABCC1 system, 044PIEZO1 gene of Er system, 045CD36 gene of CD36 system, 046ATP11C gene of ATP11C system, 047MAL gene of MAL system; Six genes were derived from 35 blood type-related genes in human platelets: ITGB3, GP1BA, ITGA2B, ITGA2, GP1BB, and CD109; Five genes were from five neutrophil systems: FCGR3B, CD177, SLC44A2, ITGAM, and ITGAL; 11 other blood type-related genes: KLF1, GATA1, SMIM1eQTL, TMEM50A, SLC35C1, PIGA, C1GALT1, C1GALT1C1, GP9, CD36, ITGB2.

3. A genotyping analysis method according to claim 1, characterized in that: All hybridization capture probes in S1 are coupled to streptavidin at the 5′ end and are evenly mixed in one tube for use.

4. A genotyping analysis method according to claim 1, characterized in that: The blood genome preparation kit in S2 is the MGIEasy blood genome DNA extraction kit.

5. A genotyping analysis method according to claim 1, characterized in that: The WGS library preparation kit used for hybridization capture in S3 to S5 is Hieff NGS One Pot Pro DNA Library Prep Kit from Yishen Biotechnology.

6. A genotyping analysis method according to claim 1, characterized in that: In the steps S7 to S9, the probe hybridization capture reagent is Nanoda DNA hybridization capture kit, the magnetic bead reagent used in the magnetic bead purification steps in S4, S5 and S9 is MGI DNA Clean Beads magnetic beads.

7. A genotyping analysis method according to claim 1, characterized in that: The library concentration determination reagent in S6 and S10 is Yisheng Bio's 1×dsDNA HS Assay Kit double-stranded DNA quantification reagent, and in S11, the MGI sequencing platform and high-throughput sequencing kit are DNBSEQ-E25RS and DNBSEQ-E25R high-throughput sequencing reagent sets.

8. A kit for use in the analysis method according to any one of claims 1 to 7, characterized in that: The invention comprises a detection kit for synchronously capturing the coding region, 5′ non-coding region and splicing site sequence of 75 blood type-related genes, and a synchronous detection kit for 63 blood type-related genes, comprising a probe set with the nucleotide sequence shown in SEQ ID NO.1 to IDNO.2023 and a 5′-end coupled streptavidin label, as well as reagents for library construction and sequencing.

9. A probe set, applied to the analysis method according to any one of claims 1 to 7, characterized in that: The invention comprises a probe set for synchronously capturing the coding region, 5' non-coding region and splicing site sequence of 75 genes, and the nucleotide sequence of the probe set is shown in SEQ ID NO.1 to ID NO.2023.

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