A set of amplification primers, an amplification system, an amplification method, a library construction method and a sequencing method for genotyping of human Lewis blood system

Abstract

This invention relates to the fields of molecular diagnostics and gene sequencing technology, specifically to a complete solution for high-precision genotyping of the human Lewis blood group system. The invention provides primer pairs for specifically amplifying long fragments of the Lewis blood group coding genes FUT2 and FUT3, and an optimized single amplification system; a library construction method adapted for nanopore sequencing; and a Lewis (FUT2, FUT3) genotyping method based on long-read data for haplotype analysis. This invention overcomes the detection limitations of conventional techniques, simultaneously acquiring the complete sequence and phase information of key regions of the FUT2 and FUT3 genes through a single amplification system, achieving accurate genotyping of common Lewis blood group phenotypes (Le(a+b−), Le(a−b+), Le(a−b−)) and effective identification of rare variants such as weakly expressed and deleted variants.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics and gene sequencing technology, specifically to a complete solution for high-precision genotyping of the human Lewis blood group system, and particularly to a method, primers, and system for simultaneous amplification, library construction, and sequencing of the full-length or key regions of the Lewis blood group coding genes FUT2 and FUT3 based on long-read nanopore sequencing technology. Background Technology

[0002] The Lewis blood group system is one of the important human erythrocyte blood group systems recognized by the International Society of Blood Transfusion (ISBT). Its antigen expression is closely correlated with an individual's secretion status, making it a key indicator for clinical transfusion safety, neonatal hemolytic disease control, and disease susceptibility risk assessment. Unlike most erythrocyte blood groups, the Lewis antigen is not synthesized by the erythrocytes themselves, but is secreted into the plasma by digestive tract epithelial cells and passively adsorbed onto the erythrocyte membrane surface in the form of soluble glycolipids. Its expression is simultaneously regulated by two genes, FUT3 (the Lewis gene) and FUT2 (the secretory gene), resulting in a complex inheritance pattern.

[0003] The FUT3 gene, located on chromosome 19, encodes α1,3 / 4-fucosyltransferase and is a core gene for Lewis antigen synthesis. Inactivation mutations in this gene directly lead to defective Lewis antigen expression. The FUT2 gene, also located on chromosome 19, encodes α1,2-fucosyltransferase and determines the secretion status of ABH substances in the human body, working synergistically with the FUT3 gene to regulate antigen phenotype. Common Lewis blood type phenotypes include Le(a+b-), Le(a-b+), Le(a+b+), and Le(ab-). The Le(ab-) phenotype is caused by a double gene defect and is recessive.

[0004] In clinical practice, although the incidence of transfusion reactions and neonatal hemolytic disease caused by Lewis blood type incompatibility is lower than that of the Rh and ABO blood group systems, the related antibodies are mostly room-temperature reactive antibodies, which can easily trigger delayed hemolytic reactions. Furthermore, the symptoms are often subtle and difficult to identify in the early stages, posing a serious threat to transfusion safety. Meanwhile, the Lewis phenotype is closely associated with various diseases. Individuals with the Lewis (ab-) phenotype have a higher susceptibility to pathogens such as Helicobacter pylori and norovirus. Additionally, the Lewis antigen is the core backbone of the tumor marker CA19-9, and abnormal expression of this antigen can directly affect the interpretation of tumor screening results. Moreover, in bone marrow and organ transplantation, Lewis blood type matching can reduce the risk of rejection and improve transplant success rates. Therefore, accurate and comprehensive Lewis blood typing has irreplaceable value for clinical transfusion, diagnosis of complex cases, and transplant matching.

[0005] Serological agglutination tests were a commonly used method for Lewis blood typing in early clinical practice, relying on anti-Lewis antibodies. a Anti-Le b Phenotypic determination is based on the agglutination reaction of specific antibodies with erythrocyte surface antigens. This method is simple to operate and low in cost, making it suitable for rapid screening at the grassroots level. However, it has several insurmountable limitations: First, Lewis antigens have strong adsorption properties and are easily affected by factors such as temperature, incubation time, and sample condition, resulting in poor stability of test results; weakly expressed antigens are prone to false negatives. Second, serological methods cannot distinguish between homozygous and heterozygous genotypes, only determining the phenotype, making it difficult to trace the genetic background. Third, for special populations such as those who have recently received blood transfusions, pregnant women, and cancer patients, the adsorption state of erythrocyte surface antigens is abnormal, easily leading to false negatives and false positives. Fourth, specific antiserum reagents are scarce and expensive, and cannot detect variant phenotypes caused by rare mutations or gene deletions, making it difficult to meet the needs of precision medicine.

[0006] To compensate for the limitations of serological methods, PCR-based molecular typing techniques have been gradually applied to Lewis blood typing. Mainstream methods include PCR sequence-specific primers (PCR-SSP), PCR restriction fragment length polymorphism (PCR-RFLP), quantitative real-time PCR, first-generation Sanger sequencing, and short-read next-generation sequencing (NGS), achieving an upgrade from phenotypic detection to genotypic detection. Conventional molecular techniques such as PCR-SSP and PCR-RFLP can only target known common mutation sites in the FUT3 and FUT2 genes, such as hotspot mutations like c.59T>G, c.314C>T, and c.385A>T in the FUT3 gene. They cannot cover rare missense mutations, nonsense mutations, splice site variations, and other complex variations. These methods are relatively cumbersome, requiring electrophoresis for interpretation, which is prone to cross-contamination and non-specific amplification. Furthermore, they cannot simultaneously analyze genotypes, easily leading to misclassification.

[0007] While first-generation Sanger sequencing can accurately detect known sites, its limited sequencing length means it can only cover gene fragments and cannot read the full-length gene. It also suffers from significant errors in detecting homologous and repetitive sequences, and its low throughput and long processing time make it unsuitable for large-scale sample screening. Short-read second-generation sequencing offers higher throughput and allows for parallel detection of multiple sites, but its read length limitation requires DNA fragmentation before sequencing and assembly. This makes it unable to fully cover the full length of FUT3 and FUT2 genes, hindering accurate identification of structural variations and fusion genes. It also cannot directly distinguish allele phases, and haplotype analysis relies on traditional blood type deduction methods with poor accuracy. Furthermore, it has significant limitations in detecting complex heterozygotes and rare alleles.

[0008] Currently, various Lewis blood typing methods struggle to simultaneously achieve accuracy, comprehensiveness, and efficiency. Conventional methods can only detect known common mutations, with limited ability to detect rare and structural variations; they cannot simultaneously perform full-length sequencing and haplotype typing of both FUT3 and FUT2 genes, easily leading to discrepancies between genotype and phenotype; some methods are cumbersome and time-consuming, failing to meet the needs of rapid typing of complex samples in clinical emergency transfusions. Currently, there is no standardized, highly targeted typing method capable of accurate sequencing of the full-length Lewis blood typing genome, precise identification of complex variations, and direct haplotype analysis, which to some extent restricts the development of precision transfusions and related disease diagnosis and treatment in clinical practice.

[0009] Chinese patent application CN120555572A utilizes microfluidic chip competitive allele-specific amplification (KASP) technology for Lewis blood group genotyping. It only detects nine pre-defined FUT2 / FUT3 gene-related single nucleotide polymorphism (SNP) sites. Because it relies solely on known sites, it is difficult to detect new gene mutations, and rare variations are easily missed.

[0010] Chinese patent application CN107630080A uses the Sanger sequencing method to determine the genotypes of Lewis blood group-related genes, targeting and amplifying the flanking sequences of FUT3 at loci 59, 202, 314, 508, and 1067, and FUT2 at finite loci 357 and 385. Due to the short sequencing read length, it is difficult to obtain complete information about the FUT2 / FUT3 genes.

[0011] Furthermore, both the KASP method and the Sanger sequencing method can only obtain the genotypes of scattered loci, making it difficult to determine the linkage relationship of variations on the same chromosome, which can easily lead to genotyping errors in compound heterozygotes.

[0012] Long-read nanopore sequencing, as a third-generation single-molecule sequencing technology, overcomes the technical limitations of short-read sequencing. With read lengths reaching tens of kb, it can completely cover the full length of FUT3 and FUT2 genes in a single reading without sequence assembly. It can directly read complete allele sequences, accurately identifying single nucleotide variants, insertions / deletions, and large structural variations, effectively solving the assembly errors and phase ambiguity problems of short-read sequencing. This technology also offers advantages such as simple library construction, fast detection speed, portable equipment, low startup cost, and real-time data analysis, making it suitable for rapid on-site clinical testing and verification of complex samples.

[0013] Currently, long-read nanopore sequencing has demonstrated excellent performance in complex blood typing systems such as Rh, MNS, and JK. However, a dedicated and standardized genotyping method has not yet been developed for the Lewis blood group system. Existing nanopore sequencing protocols are mostly general-purpose workflows that have not been optimized for the sequence characteristics, mutation hotspots, and dual-gene synergistic regulatory mechanisms of the FUT3 and FUT2 genes. These solutions suffer from low targeted enrichment efficiency, complex data analysis processes, and inconsistent typing interpretation standards, making them unsuitable for direct and accurate clinical Lewis blood typing.

[0014] The need for accurate Lewis blood typing is increasingly urgent in clinical transfusion safety, transplant matching, and disease screening. However, existing technologies have many drawbacks and cannot meet the testing needs of complex samples and rare variants. Therefore, it is necessary to develop a Lewis blood group system genotyping method based on long-read nanopore sequencing, optimize the entire process of targeted capture, library construction, and data analysis, and achieve full-length sequencing of two genes, direct haplotype analysis, and accurate identification of rare variants. This will fill the gaps in existing technologies and has significant clinical value and application prospects for improving clinical transfusion safety, perfecting the blood typing system, and contributing to the development of precision medicine. Summary of the Invention

[0015] The purpose of this invention is to address the problems of incomplete genotyping of Lewis blood group genes FUT2 and FUT3, inability to obtain phase information, and difficulty in identifying complex variants in existing technologies. This invention provides a complete solution integrating targeted synchronous amplification, library construction, and sequencing analysis. It aims to simultaneously achieve coverage and haplotype analysis of the full-length sequences of FUT2 and FUT3 genes (including all exons, introns, and expression regulatory regions) through a single amplification system, thereby improving genotyping efficiency and accuracy.

[0016] The technical solution of this invention to solve the technical problem is as follows: In a first aspect of the invention, a primer set for genotyping amplification of the human Lewis blood group system is provided, the primer set comprising a first primer pair designed for the FUT2 gene and a second primer pair designed for the FUT3 gene; The first primer pair covers a continuous region of the FUT2 gene, and the amplified fragment is approximately 10kb in size. Its upstream primer sequence is shown in SEQ ID NO: 1, and its downstream primer sequence is shown in SEQ ID NO: 2. The second primer pair covers a continuous region of the FUT3 gene, and the amplified fragment is approximately 9kb in size. Its upstream primer sequence is shown in SEQ ID NO: 3, and its downstream primer sequence is shown in SEQ ID NO: 4. The first and second primer pairs are used to simultaneously amplify the full-length sequences of FUT2 and FUT3 using a single amplification system.

[0017] The sequences of SEQ ID NO: 1-4 are as follows: .

[0018] The FUT2 gene amplification product covers the full-length sequence of the FUT2 gene (including all exons, introns, and expression regulatory regions), and the FUT3 gene amplification product covers the full-length sequence of the FUT3 gene (including all exons, introns, and expression regulatory regions).

[0019] The primer pairs consist of two pairs of forward and reverse primers designed for the FUT2 and FUT3 genes, respectively, allowing for simultaneous amplification of long fragments of both genes using a single amplification system. The FUT2 gene primer pair is designed to cover the full-length fragment of the FUT2 gene (reference sequence: NG_008107.1) from upstream of the transcription start site to the 3'UTR region, covering its entire sequence (including all exons, introns, and expression regulatory regions). The FUT3 gene primer pair is designed to cover the full-length fragment of the FUT3 gene (reference sequence: NG_008108.1) from upstream of the transcription start site to the 3'UTR region, covering its entire sequence (including all exons, introns, and expression regulatory regions). The primers are 30 bp in length, have a GC content of 45%-55%, and a Tm value of 60-65℃.

[0020] In a second aspect of the invention, a gene typing amplification system for the human Lewis blood group system is provided, which is a single amplification system for simultaneously amplifying long fragments of the Lewis coding genes FUT2 and FUT3.

[0021] The amplification system comprises: a PCR premix, the FUT2 and FUT3 amplification primer pairs as described in the first aspect, and template DNA (human genomic DNA). The PCR premix contains a high-fidelity long-fragment DNA polymerase KeyPo SE DNAPolymerase, dNTPs, and Mg. 2+ Essential components for PCR reactions.

[0022] The total volume of the system is 24 μL, and the final concentrations of each component are: 0.2-0.5 μM for each primer, 100-200 ng for template DNA, and the remaining components are configured according to the conventional long-fragment PCR system. This system can simultaneously achieve efficient amplification of long fragments of FUT2 and FUT3 genes without the need to set up two separate amplification systems.

[0023] In a third aspect of the invention, a method is provided for simultaneously amplifying FUT2 and FUT3 using primer pairs as described in the first aspect or a single amplification system as described in the second aspect.

[0024] The method includes the following steps: 1) Extract human genomic DNA.

[0025] 2) Prepare a single amplification system as described in the second aspect by adding FUT2 and FUT3 primer pairs; there is no need to prepare the system separately. 3) Run the optimized long-fragment PCR program: pre-denaturation at 94℃ for 2 minutes; followed by denaturation at 98℃ for 10 seconds, annealing at 62℃ for 30 seconds, and extension at 68℃ for 5 minutes (adapting to the length of the two gene amplification products, no need to adjust the extension time separately), cycle 30-35 times; finally store at 4℃ to obtain the long-fragment amplification products of FUT2 and FUT3 genes simultaneously.

[0026] In a fourth aspect of the present invention, a method for constructing libraries of FUT2 and FUT3 gene amplification products based on nanopore sequencing is provided.

[0027] The method described herein, for the long fragment products of FUT2 and FUT3 genes obtained by simultaneous amplification as described in the third aspect, employs the following steps to construct a library directly adapted for nanopore sequencing: 1) Purification and quantification of long fragment amplification products: PCR products were purified using magnetic beads (simultaneous purification of amplification products of two genes), and the concentration was determined using a Qubit nucleic acid quantification instrument; 2) End repair: Long fragment amplification and purification products are processed with end repair enzymes and DNA repair enzymes to unify the end structure and ensure efficient subsequent adapter ligation. 3) Sequencing adapter ligation: Using barcode adapters with different tags corresponding to the nanopore sequencer and sequencing adapters, the adapters are ligated to the end repair products of different samples using ligase; 4) Library purification and quality control: The final library was purified using magnetic beads and the concentration was detected by a Qubit nucleic acid quantification instrument to obtain a barcode-enabled library that can be used for nanopore sequencing.

[0028] In a fifth aspect of the invention, a method for sequencing FUT2 and FUT3 genes based on nanopore sequencing technology is provided.

[0029] The method includes the following steps: 1) The library constructed as described in the fourth aspect is mixed with sequencing reaction buffer and loaded into the sequencing chip of a nanopore sequencer; 2) Start the sequencing software and set the corresponding sequencing duration and data volume threshold; 3) Real-time base identification is performed to generate raw sequence data in FASTQ format, and long-read sequence data of FUT2 and FUT3 genes are obtained simultaneously. The data is split back to the corresponding samples according to the barcode connectors of different labels.

[0030] In a sixth aspect of the present invention, a method for FUT2 and FUT3 genotyping and haplotype analysis is provided.

[0031] The method described is based on read length data obtained by sequencing methods such as the fifth aspect, and includes the following steps: 1) Align the high-quality read data to the reference sequences of the FUT2 and FUT3 genes, respectively; 2) Identify all SNPs, including hotspot SNPs, and Indel variants in the FUT2 and FUT3 genes, respectively; 3) Taking advantage of long read lengths, haplotypes (phases) of FUT2 and FUT3 gene variations can be determined directly based on the linkage relationships of polymorphic sites in the read length data. 4) Based on the known FUT2 and FUT3 allele databases, determine the final Lewis genotype according to the haplotype combination of the two genes, including Le(a+b-), Le(a-b+), Le(ab-) and rare alleles, and perform functional prediction on new variants.

[0032] In a seventh aspect of the invention, the use of the primer set as described in the first aspect or the amplification system as described in the second aspect is provided in the preparation of kits or systems for accurate Lewis blood typing, prediction of hemolytic transfusion reaction risk, and identification of rare Lewis blood types.

[0033] The present invention has the following technical effects: 1) Comprehensive and accurate typing: The FUT2 and FUT3 genes can be simultaneously detected in all coding regions and key non-coding regions through a single amplification system. It can not only accurately distinguish common Lewis blood group phenotypes (Le(a+b-), Le(a-b+), Le(ab-)), but also effectively identify rare genotypes such as weak expression and deletion, thus solving difficult serological typing problems.

[0034] 2) Direct haplotype analysis: Long-read sequencing can directly obtain linkage information of each variant site of the FUT2 and FUT3 genes on the same chromosome, clarify the phase, which is crucial for the accurate typing of compound heterozygotes and avoids the phase judgment error of short-read sequencing.

[0035] 3) High efficiency and convenience: The FUT2 and FUT3 genes are amplified simultaneously using a single amplification system, eliminating the need to set up two separate amplification systems. This simplifies the operation process, shortens the detection time, reduces operational errors and experimental costs, and is more suitable for routine molecular typing in clinical laboratories.

[0036] 4) High sensitivity and specificity: Targeted amplification improves the sequencing depth of the target regions of FUT2 and FUT3 genes. Combined with high-fidelity enzymes and optimized systems, it ensures the sensitivity and specificity of detection, and is especially suitable for the analysis of low-frequency variants and rare alleles.

[0037] 5) Discovering new allele potential: Full-length sequencing of FUT2 and FUT3 genes helps to discover and characterize new alleles, promotes in-depth research on the Lewis blood group system, and enriches the Lewis blood group gene polymorphism database. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the FUT2 and FUT3 gene structures and the locations of the amplification primers in Example 1.

[0039] Figure 2 This is an agarose gel electrophoresis image of the simultaneous amplification of long fragments of the FUT2 and FUT3 genes in a single amplification system in Example 2 (showing a single bright band of the expected size).

[0040] Figure 3 This is an interface diagram showing the setting of experimental parameters for the nanopore sequencer in Embodiment 4 of the present invention.

[0041] Figure 4 This is an example diagram of haplotype analysis of FUT2 and FUT3 genes using long read data in Embodiment 4 of the present invention, showing multiple SNP sites covered on the read data. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0043] The main raw materials involved in this invention are listed below: .

[0044] Example 1: Design and Synthesis of Primers for Long Fragment Amplification of Lewis (FUT2, FUT3) Genes For the FUT2 gene (NG_008107.1) and the FUT3 gene (NG_008108.1), two pairs of long-fragment amplification primers (e.g., 1 pair) covering all exons and flanking introns were designed respectively. Figure 1 As shown in the figure, the primer pair can simultaneously amplify long fragments of two genes through a single amplification system.

[0045] The sequences of the two pairs of long fragment amplification primers are shown in SEQ ID NO: 1-4, and the specific sequences are shown in Table 1: .

[0046] The primers were synthesized and purified by a synthetic company. The primer powder was dissolved and diluted with TE (10 mmol / L Tris-HCl 0.1 mmol / L EDTA) to a working concentration of 12 OD.

[0047] The preparation of the amplification primer mixture (a single mixture containing FUT2 and FUT3 primer pairs) is shown in Table 2.

[0048] Table 2: Preparation of amplification primer mixture .

[0049] Example 2: Synchronous amplification of long fragments of Lewis (FUT2, FUT3) genes In this embodiment, the method for simultaneous amplification of long fragments of the Lewis (FUT2, FUT3) gene includes the following steps: 1) Extracting human genomic DNA; 2) Prepare a single amplification system by adding FUT2 and FUT3 primer pairs; there is no need to prepare the system separately. 3) Run the PCR program to simultaneously obtain long fragment amplification products of FUT2 and FUT3 genes.

[0050] In this embodiment, the steps for nucleic acid sample extraction include: Blood samples can be collected using blood collection tubes containing sodium citrate or ethylenediaminetetraacetic acid (EDTA) as anticoagulants. The experimental samples must be fresh or frozen whole blood samples that have not undergone repeated freeze-thaw cycles. In this example, EDTA-anticoagulated whole blood is used.

[0051] Nucleic acid extraction can be performed using precipitation, column chromatography, or magnetic bead methods to obtain sufficient quantity and quality of nucleic acid for long-fragment amplification reactions. In this embodiment, column chromatography is used.

[0052] The concentration of extracted nucleic acid samples should be controlled between 20-40 ng / μL, and the A260 / A280 ratio should be between 1.6 and 2.0. In this example, the nucleic acid sample concentration is uniformly 30 ng / μL.

[0053] In this embodiment, the Lewis (FUT2, FUT3) long fragment amplification reaction system adopts a single amplification system to simultaneously amplify the FUT2 and FUT3 genes. The amplification system is shown in Table 3.

[0054] Table 3: Amplification System .

[0055] The PCR premix (PK512) in Table 3 is a PCR reaction solution containing high-fidelity DNA polymerase, dNTPs, and Mg2+, which are essential components for amplification.

[0056] In this embodiment, the steps for simultaneous amplification of long fragments of the Lewis (FUT2, FUT3) genes are as follows: The single amplification system was amplified to simultaneously obtain FUT2 and FUT3 gene amplification products. The amplification parameters were as follows: 94℃, pre-denaturation for 2 minutes; 98℃, denaturation for 10 seconds; 62℃, annealing for 30 seconds; 68℃, extension for 5 minutes (to accommodate a product length of approximately 10kb), 30 cycles; storage at 4℃. The specific amplification reaction program is shown in Table 4.

[0057] Table 4: Amplification Reaction Procedure .

[0058] In this embodiment, the steps for mass control of amplification products include: The products of the long-fragment amplification reaction were analyzed using 1% agarose gel electrophoresis to determine whether the FUT2 and FUT3 genes were amplified synchronously. Voltage: 220V, Time: 20 minutes; the amplification products were confirmed by electrophoresis to be 10kb (FUT2) and 9kb (FUT3) respectively. After the absence of specific bands (such as...), the amplification products were analyzed. Figure 2 As shown in the figure, the amplified products are prepared for library construction.

[0059] Example 3: Construction of Nanopore Sequencing Libraries Nanopore sequencing library construction includes the following steps: 1) Purification and quantification of long fragment amplification products: PCR products were purified using magnetic beads (i.e., the simultaneous purification of two gene amplification products in Example 2), and the concentration was determined using a Qubit nucleic acid quantification instrument; 2) End repair: Long fragment amplification and purification products are processed with end repair enzymes and DNA repair enzymes to unify the end structure and ensure efficient subsequent adapter ligation. 3) Sequencing adapter ligation: Using barcode adapters with different tags corresponding to the nanopore sequencer and sequencing adapters, the adapters are ligated to the end repair products of different samples using ligase; 4) Library purification and quality control: The final library was purified using magnetic beads and the concentration was detected by a Qubit nucleic acid quantification instrument to obtain a barcode-enabled library that can be used for nanopore sequencing.

[0060] The specific steps are explained below: First, purify the long fragment amplification product, as follows: 1) Simultaneous purification of long fragment amplification products of the FUT2 and FUT3 genes obtained by synchronous amplification was performed using purified magnetic beads. First, the purified magnetic beads were equilibrated at room temperature, followed by vortexing to mix. Quality-controlled long fragment amplification products were selected and placed in centrifuge tubes, with purified magnetic beads added at a 1:1 ratio to sample volume.

[0061] 2) Gently tap the centrifuge tube to mix thoroughly, and let it stand at room temperature for 5 minutes to allow the DNA to fully bind to the magnetic beads. Then, place the centrifuge tube on a magnetic rack and let it stand at room temperature for 2 minutes until the magnetic beads are completely adsorbed. Carefully aspirate and discard the supernatant.

[0062] 3) Keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, wash the surface of the magnetic beads, let stand for 30 seconds, then remove and discard the ethanol; repeat the above washing steps once. Open the centrifuge tube cap and let stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely.

[0063] 4) Remove the centrifuge tube from the magnetic rack, add 25 μL of nuclease-free water, gently tap to mix, and let stand at room temperature for 5 minutes.

[0064] 5) Place the centrifuge tube back on the magnetic rack and let it stand at room temperature for 2 minutes until the magnetic beads are completely adsorbed. Then carefully aspirate the supernatant into a new centrifuge tube.

[0065] 6) Use the Qubit nucleic acid quantification instrument to determine the concentration of the purified amplified product.

[0066] Then, the long fragment amplified and purified products were processed using end-repair enzymes and DNA repair enzymes to standardize the end structure and ensure efficient subsequent adapter ligation. The specific steps for end repair are as follows: 500 ng of purified amplified product (containing FUT2 and FUT3 gene products) was added to a centrifuge tube, and the purified amplified product was subjected to end repair. The end repair system is shown in Table 5. The repair procedure is shown in Table 6.

[0067] Table 5: End-of-Life Remediation Systems .

[0068] Table 6: Repair Procedure .

[0069] After end repair, multiple sample libraries were constructed using the corresponding barcode adapters and sequencing adapters for the nanopore sequencer.

[0070] The steps for ligating specific barcode adapters to different samples include: first, preparing the barcode ligation reaction system; then, using the corresponding barcode adapter for the nanopore sequencer, ligating the barcode adapter to the end repair product using a ligase.

[0071] The barcode-linked reaction system is shown in Table 7.

[0072] Table 7: Barcode-linked reaction system: .

[0073] After the reaction system is prepared, gently tap to mix, briefly centrifuge, and let it react at room temperature for 10 minutes. After the reaction is complete, add 2 μL of termination buffer to terminate the reaction, and mix equal volumes of samples with different barcodes.

[0074] After connecting the barcode connector, purify the sample mixed library connected to the barcode connector. The purification steps are as follows: 1) Add the purification magnetic beads at a ratio of 1:0.4, gently tap to mix, and let stand at room temperature for 5 minutes; after instantaneous centrifugation, place on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0075] 2) Add 500 μL of 80% ethanol to wash, and discard the supernatant after 30 seconds; repeat the above washing steps once. Open the tube cap and let it stand for 5 minutes until the surface of the magnetic beads is dull and the residual ethanol has completely evaporated.

[0076] 3) Add 50 μL of nuclease-free water to resuspend the magnetic beads and let stand at room temperature for 3 minutes.

[0077] 4) After instantaneous centrifugation, place the centrifuge tube on a magnetic rack and wait for the solution to clarify before transferring the supernatant to a new centrifuge tube.

[0078] 5) Add 1 volume of purified magnetic beads, gently tap to mix, and let stand at room temperature for 5 minutes; centrifuge briefly, place the centrifuge tube on a magnetic rack, and let stand for 2 minutes after the solution becomes clear, until the magnetic beads are completely adsorbed, then discard the supernatant.

[0079] 6) Add 200uL of 80% ethanol, let stand for 30 seconds and then aspirate the supernatant; repeat once; after instantaneous centrifugation, completely aspirate the remaining liquid in the tube, open the cap and let stand for 2-3 minutes to allow the residual ethanol to evaporate completely.

[0080] 7) Remove the centrifuge tube from the magnetic rack, add 35 μL of nuclease-free water to resuspend the magnetic beads, and let stand at room temperature for 3 minutes.

[0081] 8) After instantaneous centrifugation, place the centrifuge tube on a magnetic rack. After the magnetic beads are completely adsorbed, transfer the supernatant to a new centrifuge tube.

[0082] 9) Use the Qubit nucleic acid quantification instrument to determine the concentration of the purified barcode-linked sample mixed library.

[0083] Next, the sequencing adapter ligation reaction will be performed.

[0084] The sequencing adapter ligation reaction system is shown in Table 8.

[0085] .

[0086] The steps for ligating sequencing adapters to the mixed library are as follows: After adding the sample, gently tap to mix, briefly centrifuge, and incubate at room temperature for 10 minutes to allow the sequencing adapters to fully ligate with the DNA fragments in the mixed library.

[0087] Finally, the final library was purified using magnetic beads, as follows: 1) Add the purification magnetic beads at a ratio of 1:0.4, gently tap to mix, and let stand at room temperature for 5 minutes; after instantaneous centrifugation, place on a magnetic rack, and discard the supernatant after the solution becomes clear.

[0088] 2) Remove the centrifuge tube from the magnetic rack, add 200 μL of room temperature equilibrated long fragment washing buffer, gently tap the tube wall to resuspend the magnetic beads, centrifuge briefly, then place the centrifuge tube back on the magnetic rack. After the magnetic beads are completely adsorbed, discard the supernatant. Repeat the above washing steps once.

[0089] 3) Aspirate as much residual liquid as possible from the centrifuge tube, immediately resuspend the magnetic beads in 16 μL of elution buffer (AEB), and let stand at room temperature for 5 minutes.

[0090] 4) After instantaneous centrifugation, place the centrifuge tube back on the magnetic rack and wait for the magnetic beads to be fully attracted before transferring the supernatant to a new centrifuge tube.

[0091] 5) After purification, the product concentration is measured using a Qubit nucleic acid quantification instrument, which is the final sequencing library.

[0092] Example 4: Nanopore Sequencing and Data Analysis In this embodiment, the steps for configuring the sequencing system and performing sequencing include: Mix 35 fmol of the final library with the sequencing reaction buffer and load it into the sequencing chip of the nanopore sequencer.

[0093] like Figure 3 The sequencer's onboard parameter settings interface is shown. Based on specific sequencing needs, parameters such as sequencing time, data volume threshold, and quality threshold (quality value > 7) can be set. The sequencing software is then run, and the nanopore sequencer performs real-time barcode decoding and base recognition, generating raw sequence data in FASTQ format. Simultaneously, long-read sequence data for the FUT2 and FUT3 genes are obtained. The data is then separated back into the corresponding samples according to the barcode adapters with different labels.

[0094] In this embodiment, the steps for analyzing sequencing data include: 1) Sequence alignment and variant identification: The sequencing read data were aligned to the reference sequences of FUT2 and FUT3 genes using analysis software, and variant identification of the two genes was performed through the alignment.

[0095] 2) Haplotype typing and result interpretation: Linkage is determined directly from read length data covering each variant site in the FUT2 and FUT3 genes. Figure 4 Based on the identified FUT2 and FUT3 gene haplotype combinations, the final Lewis genotype was determined with reference to the ISBT database. The results are shown in Table 9.

[0096] Table 9: Lewis blood group genotyping results of the samples .

[0097] The results demonstrate that this method successfully identified the genotypes corresponding to all known Lewis blood group phenotypes, and the typing accuracy was higher than that of conventional methods.

[0098] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A primer set for genotyping and amplification of the human Lewis blood group system, characterized in that, The primer set includes a first primer pair designed for the FUT2 gene and a second primer pair designed for the FUT3 gene; The first primer pair has an upstream primer sequence as shown in SEQ ID NO: 1 and a downstream primer sequence as shown in SEQ ID NO: 2; The second primer pair has the upstream primer sequence shown in SEQ ID NO: 3 and the downstream primer sequence shown in SEQ ID NO: 4; The primer pairs are used to amplify the Lewis blood group coding genes FUT2 and FUT3 for long-read sequencing, and the primer pairs are amplified simultaneously using a single amplification system.

2. A gene typing and amplification system for the human Lewis blood group system, characterized in that, It is a single amplification system for simultaneously amplifying long fragments of the Lewis coding genes FUT2 and FUT3, comprising the primer pair as described in claim 1, as well as PCR premix and template DNA.

3. A method for simultaneously amplifying FUT2 and FUT3 using the primer pair as described in claim 1 or the amplification system as described in claim 2, characterized in that, The method includes the following steps: 1) Extracting human genomic DNA; 2) To prepare the single amplification system as described in claim 2, add the FUT2 and FUT3 primer pairs; there is no need to prepare the system separately. 3) Run the PCR program: pre-denaturation at 94℃ for 2 minutes; then denaturation at 98℃ for 10 seconds, annealing at 62℃ for 30 seconds, extension at 68℃ for 5 minutes, cycle 30-35 times; finally store at 4℃ to obtain long fragment amplification products of FUT2 and FUT3 genes simultaneously.

4. A method for constructing libraries of FUT2 and FUT3 gene amplification products based on nanopore sequencing, characterized in that, The method described herein is for the simultaneous amplification of long fragment products of the FUT2 and FUT3 genes obtained by the method described in claim 3. The following steps are used to construct a library directly adapted for nanopore sequencing: 1) Purification and quantification of long fragment amplification products: PCR products were purified using magnetic beads and their concentrations were determined using a Qubit nucleic acid quantification instrument; 2) End repair: The long fragment amplification and purification products are processed using end repair enzymes and DNA repair enzymes; 3) Connect the nanopore sequencing barcode adapter and the sequencing adapter; 4) Purify to obtain a barcode-encoded library that can be used for nanopore sequencing.

5. A method for sequencing FUT2 and FUT3 genes based on nanopore sequencing technology, characterized in that, The method includes the following steps: 1) The library constructed by the method described in claim 4 is mixed with sequencing reaction buffer and loaded into the sequencing chip of a nanopore sequencer; 2) Start the sequencing software and set the corresponding sequencing duration and data volume threshold; 3) Real-time base recognition is performed to generate raw sequence data in FASTQ format, and long-read sequence data of FUT2 and FUT3 genes are obtained simultaneously.

6. A method for FUT2 and FUT3 genotyping and haplotype analysis, characterized in that, The method, based on read data obtained by the sequencing method as described in claim 5, includes the following steps: 1) Align the high-quality read data to the reference sequences of the FUT2 and FUT3 genes, respectively; 2) Identify all SNPs, including hotspot SNPs, and Indel variants in the FUT2 and FUT3 genes, respectively; 3) Taking advantage of long read lengths, haplotypes of FUT2 and FUT3 gene variations can be determined directly based on the linkage relationships of polymorphic sites in the read length data; 4) Based on the known FUT2 and FUT3 allele databases, determine the final Lewis genotype according to the haplotype combination of the two genes, including Le(a+b-), Le(a-b+), Le(ab-) and rare alleles, and perform functional prediction on new variants.

7. The application of the primer set as described in claim 1 or the amplification system as described in claim 2 in the preparation of kits or systems for accurate Lewis blood typing, prediction of hemolytic transfusion reaction risk, and identification of rare Lewis blood types.

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