A kind of amplification primer set, amplification system, amplification method, library construction method and sequencing method for Kidd blood group system genotyping based on long-read nanopore sequencing

Abstract

This invention relates to the fields of molecular diagnostics and gene sequencing technology, specifically to a primer set, amplification system, amplification method, library construction method, and sequencing method for Kidd blood group system genotyping amplification based on long-read nanopore sequencing. This invention provides primers and an optimized amplification system for specifically amplifying long fragments of the Kidd blood group encoding gene SLC14A1; a library construction method adapted for nanopore sequencing; and a SLC14A1 genotyping method based on long-read data for haplotype analysis. This invention overcomes the limitations of conventional techniques that only detect single SNPs, enabling the simultaneous acquisition of the complete sequence and phase information of key regions of the SLC14A1 gene, achieving accurate genotyping of JKA / JKB and effective identification of rare variants such as weakly expressed and deleted variants. This invention provides a powerful molecular tool for clinical transfusion safety assurance, rare blood group bank construction, and genetic research.

Description

Technical Field

[0001] 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 Kidd blood group system, particularly a primer set, amplification system, amplification method, library preparation method, and sequencing method for Kidd blood group system genotyping amplification based on long-read nanopore sequencing. Background Technology

[0002] The Kidd blood group system, one of the important red blood cell blood group systems in humans, is designated ISBT009 by the International Society of Blood Transfusion (ISBT). The symbol for the Kidd blood group system in ISBT nomenclature is JK, so it can also be called the JK blood group system. The coding gene for the Kidd blood group system, SLC14A1, is located on chromosome 18q11-12. This gene encodes the Kidd glycoprotein, which also functions as a urea transporter, participating in the active transport of urea across the red blood cell membrane. The Kidd blood group system mainly encompasses the JK... a 、Jk b The three antigens Jk1, Jk2, and Jk3 correspond to four phenotypes: Jk(a+b+), Jk(a+b-), Jk(a-b+), and Jk(ab-). Among them, Jk... a With Jk b The antigen is encoded by the JKA and JKB alleles formed by a single base substitution (c.838G>A) at position 838 of the SLC14A1 gene. The Jk3 antigen is encoded by the Jk allele. a With Jk b Common epitopes of antigens are widely present in the vast majority of the population.

[0003] In the field of clinical transfusion medicine, accurate typing of the Kidd blood group system is of paramount importance. When Jk antibodies produced by the body's immune system bind to their corresponding Jk antigens, they activate the complement system, leading to the destruction of red blood cells and triggering severe hemolytic transfusion reactions, especially delayed-type hemolytic transfusion reactions. Furthermore, Kidd blood type incompatibility between mother and child can also lead to hemolytic disease of the newborn, posing a serious threat to the patient's life. Therefore, accurate Kidd blood group genotyping is a crucial prerequisite for ensuring clinical transfusion safety and achieving maternal and infant health. In addition, with the development of transfusion medicine, the need for screening and identifying rare Kidd blood group variants is becoming increasingly urgent. These variants are often caused by rare mutations, deletions, or splicing site abnormalities in the SLC14A1 gene, and accurate typing of these variants is of great value for precise transfusion of specific patients.

[0004] Currently, the typing methods for the Kidd blood group system are mainly divided into two categories: traditional serological testing and molecular biological genotyping. Among them, traditional serological methods are widely used in clinical practice due to their simplicity and low cost, but they have significant limitations: firstly, the detection rate of Jk antibodies is extremely low, and related studies have shown that Jk... a 、Jk b The correct detection rate of antibodies is less than 30%, and is easily missed or misdetected due to factors such as the characteristics of Jk antibodies, dose effect and complement dependence. On the other hand, serological methods are difficult to accurately classify patients who have recently received blood transfusions or have received repeated blood transfusions, and the specific antiserum used is expensive. At the same time, it is impossible to distinguish the phenotype corresponding to heterozygous genotypes, and the interpretation criteria are difficult to unify, which poses serious risks to clinical blood transfusion safety and cannot meet the needs of accurate classification.

[0005] To overcome the limitations of serological methods, molecular biology genotyping methods are increasingly being applied to Kidd blood typing. Currently used techniques include PCR sequence-specific primers (PCR-SSP), Sanger sequencing, and next-generation sequencing (NGS). Among these, PCR-SSP requires gel electrophoresis analysis, and its multi-step operation easily introduces inter-well errors and cross-contamination, resulting in relatively low detection efficiency. Furthermore, conventional molecular typing techniques (such as PCR-SSP and Sanger sequencing) typically only detect the c.838G>A (p.Asp280Asn) single nucleotide polymorphism (SNP) in exon 8 of the SLC14A1 gene to distinguish between the JKA and JKB alleles. However, the SLC14A1 gene is approximately 30kb in length and contains 10 exons; its phenotypic complexity stems not only from c.838G>A but may also be related to variations in other exons, introns, or regulatory regions.

[0006] Next-generation sequencing (NGS) technology, as a commonly used high-throughput genotyping method, enables simultaneous detection of multiple samples and multiple loci. Some molecular probe-capture-based NGS methods have been applied to Kidd blood typing, effectively detecting JKA and JKB alleles. However, this technology still has inherent limitations. NGS is a short-read sequencing method, requiring the fragmentation of genomic DNA before sequencing, making it particularly difficult to accurately identify structural variations such as repetitive sequences, large deletions, insertions, and gene recombination. Furthermore, short-read sequencing cannot obtain complete haplotype information, making it difficult to distinguish allele phase relationships. For complex genotypes formed by combinations of multiple variant sites, genotyping errors are prone to occur. Additionally, the sequencing process is complex and library construction is time-consuming, making it difficult to meet the needs of rapid clinical testing.

[0007] Long-read nanopore sequencing, as a core representative of third-generation sequencing technology, has shown broad application prospects in the field of genotyping due to its unique technological advantages. This technology uses nanopore arrays to detect changes in current as DNA molecules pass through, enabling real-time interpretation of DNA sequences. It eliminates the need for DNA fragmentation, directly obtaining ultra-long read sequence information and fully covering the entire SLC14A1 gene. This technology effectively avoids the splicing errors of short-read sequencing. Furthermore, it features compact instrument size, flexible deployment, low sequencing cost, rapid data acquisition, and low startup cost. It enables real-time data analysis and can simultaneously detect single nucleotide variants and structural variations, accurately resolving haplotype information, making it particularly suitable for the screening and identification of rare variants.

[0008] Chinese patent 201510125425.X discloses a primer set and kit for detecting Kidd blood type genotyping of human red blood cells. The PCR sequence-specific primer (PCR-SSP) method only targets the c.838G>A site of exon 8 for amplification and electrophoresis interpretation. It can only distinguish the conventional JKA / JKB genotype and cannot detect other variants and haplotypes. The method is more limited overall.

[0009] Long-read sequencing methods cover 10–20kb per read, enabling the acquisition of complete sequences and haplotype phase information in a single run. They can simultaneously detect single nucleotide mutations (SNPs), large deletions / insertions, splice site variations, structural variations, and other variant types. They can accurately identify JKA / JKB and rare variants such as weakly expressed and deleted variants, comprehensively solving the problems of incomplete typing, inability to determine phases, and difficulty in identifying complex variants in traditional methods.

[0010] In summary, existing Kidd blood typing methods all have certain limitations and cannot meet the clinical needs for accurate, rapid, and comprehensive typing of the Kidd blood group system, especially in the areas of rare variant screening and complex genotyping. Therefore, developing a genotyping method based on long-read nanopore sequencing technology and optimized for the Kidd blood group system to overcome the shortcomings of existing technologies, achieve accurate and efficient Kidd blood typing, and ensure clinical transfusion safety has become an urgent technical challenge in the field of transfusion medicine. Summary of the Invention

[0011] The purpose of this invention is to address the problems of incomplete genotyping, inability to obtain phase information, and difficulty in identifying complex variants in existing technologies for the Kidd blood group coding gene SLC14A1. This invention provides a complete solution integrating targeted amplification, library construction, and sequencing analysis, aiming to achieve coverage of the full-length sequence (including all exons, introns, and expression regulatory regions) of the Kidd blood group coding gene SLC14A1 and haplotype analysis.

[0012] 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 Kidd blood group system based on long-read nanopore sequencing is provided. The primer set includes primer pairs for amplifying the Kidd blood group coding gene SLC14A1 for long-read sequencing, covering a continuous region of the SLC14A1 gene, wherein the continuous region includes all exons, introns and expression regulatory regions.

[0013] The primer set consists of a first primer pair and a second primer pair: The first primer pair, code JK01, amplifies a fragment of approximately 10kb. 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, designated JK02, amplifies a fragment of approximately 20 kb. Its upstream primer sequence is shown in SEQ ID NO: 3, and its downstream primer sequence is shown in SEQ ID NO: 4.

[0014] The primer pairs are designed to cover the full-length fragment of the SLC14A1 gene (reference sequence: NG_011747.1) from upstream of the transcription start site to the 3'UTR region, encompassing all exons, introns, and expression regulatory regions. The primers are 30 bp in length, with a GC content of 40–60% and a Tm value of 60–70 °C. The amplified product is 10–20 KB in length to cover a contiguous region containing multiple exons.

[0015] The specific sequences of the primer pairs are as follows:

[0016] In a second aspect of the invention, a genotyping and amplification system for the Kidd blood group system based on long-read nanopore sequencing is provided.

[0017] The system comprises: a PCR premix, primer pairs as described in the first aspect, and template DNA (human genomic DNA). The PCR premix comprises high-fidelity long-fragment DNA polymerase (KeyPo SE DNA Polymerase), dNTPs, and Mg... 2+ Essential components for PCR reactions, such as PCR buffer.

[0018] 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 and 100-200 ng for template DNA.

[0019] In a third aspect of the invention, a method for amplifying the SLC14A1 gene using primer pairs as described in the first aspect or a system as described in the second aspect is provided.

[0020] The method includes the following steps: 1) Extracting human genomic DNA; 2) Prepare the amplification system as described in the second aspect; 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, extension at 68℃ for 5-10 minutes (adjust according to product length), cycle 30-35 times; finally store at 4℃.

[0021] In a preferred embodiment of the present invention, the amplification system for the long fragment of the SLC14A1 gene includes a first amplification system and a second amplification system; The first amplification system is as follows:

[0022] The second amplification system is as follows:

[0023] In a preferred embodiment of the present invention, the long fragment amplification step of the SLC14A1 gene is specifically as follows: The first amplification system was amplified to obtain the first amplification product. 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 10 kb), 30 cycles; storage at 4℃. The second amplification system was amplified to obtain the second amplification product. 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 10 minutes (to accommodate a product length of approximately 20 kb), 30 cycles; storage at 4℃.

[0024] The first and second amplification products were mixed in equimolar amounts and used as samples for constructing nanopore sequencing libraries.

[0025] In a fourth aspect of the present invention, a method for constructing a library of SLC14A1 gene amplification products based on nanopore sequencing is provided.

[0026] The method described herein, for long-fragment amplification products obtained as 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 and their concentrations were 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 and sequencing adapters with different tags corresponding to the nanopore sequencer, the adapters are ligated to the end repair products of different samples using ligase; the barcode adapters tag different samples for subsequent data splitting; the sequencing adapters provide chip binding sites + primer binding sites. Without adapters, the sequencing instrument cannot start the sequencing reaction.

[0027] 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 present invention, a method for sequencing the SLC14A1 gene 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 the SLC14A1 gene is obtained. The data is then 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 SLC14A1 genotyping and haplotype analysis is provided: Based on the read length data obtained by sequencing methods such as the fifth aspect, the following steps are taken: 1) Align high-quality read data to the SLC14A1 gene reference sequence; 2) Identify all SNPs and Indel variants, including c.838G>A; 3) Taking advantage of long read lengths, haplotypes (phases) can be determined directly based on the linkage relationships of polymorphic sites on read length data, using multiple variant sites covered on a single read length. 4) Based on the known SLC14A1 allele database, determine the alleles, including JKA, JKB and rare alleles, and perform functional prediction on new variants.

[0031] 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 Kidd blood typing, prediction of hemolytic transfusion reaction risk, and identification of rare Kidd blood types.

[0032] The present invention has the following technical effects: 1) Comprehensive and accurate typing: It can detect all coding regions and key non-coding regions of the Kidd blood type coding gene SLC14A1 in one go. It can not only accurately distinguish JKA / JKB, but also effectively identify weak expression type, deletion type and rare recombinant type, solving the problem of difficult serological typing.

[0033] 2) Direct haplotype analysis: Long-read sequencing can directly obtain linkage information of c.838G>A and other potential variant sites on the same chromosome, clarifying the phase, which is crucial for the accurate typing of compound heterozygotes.

[0034] 3) High sensitivity and specificity: Targeted amplification improves the sequencing depth of the target region. Combined with high-fidelity enzymes and optimized systems, it ensures the sensitivity and specificity of detection, and is especially suitable for low-frequency variant analysis.

[0035] 4) Integrated and efficient process: This invention provides a standardized process from sample to report, which greatly reduces the amount of data, analysis complexity and cost, and is more suitable for routine molecular typing in clinical laboratories.

[0036] 5) Discovering new allele potential: Full-length sequencing of the Kidd blood group coding gene SLC14A1 helps to discover and characterize new alleles, and promotes in-depth research on the Kidd blood group system. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the gene structure and amplification primer design location of the Kidd blood type encoding gene SLC14A1 in Example 1.

[0038] Figure 2 The image shows an agarose gel electrophoresis of the long fragment PCR product of the Kidd blood type encoding gene SLC14A1 in Example 2, displaying a single bright band of the expected size.

[0039] Figure 3 This is a diagram of the interface for setting the experimental parameters on the nanopore sequencer in Example 4.

[0040] Figure 4 This is an example diagram of haplotype analysis of the Kidd blood group coding gene SLC14A1 using long read data, as shown in Example 4. It illustrates multiple SNP sites covered on the read data. Detailed Implementation

[0041] 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.

[0042] The main raw materials involved in this invention are listed below:

[0043] Example 1: Design and synthesis of primers for long fragment amplification of the Kidd blood type coding gene SLC14A1 Two pairs of long-fragment amplification primers (e.g., NG_011747.1) covering exons 1 to 10 and flanking introns were designed to target the Kidd blood group coding gene (SLC14A1, NG_011747.1). Figure 1 (As shown).

[0044] The sequences of the two pairs of long fragment amplification primers are shown in SEQ ID NO: 1-SEQ ID NO: 4, as detailed in Table 1.

[0045]

[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 120 D.

[0047] Prepare primer mixtures for two amplification primer pairs, as shown in Table 2.

[0048]

[0049] Example 2: Amplification of a long fragment of the SLC14A1 gene The SLC14A1 gene amplification method of this embodiment includes the following steps: 1) Extracting human genomic DNA; 2) Prepare the amplification system; 3) Run the long fragment PCR program to amplify the long fragment of the SLC14A1 gene.

[0050] In this embodiment, the method for extracting nucleic acid samples includes the following steps: 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 amplification system for the long fragment of the SLC14A1 gene includes a first amplification system and a second amplification system. The components of the first amplification system are shown in Table 3, and the components of the second amplification system are shown in Table 4.

[0054] Table 3: First Amplification System

[0055] Table 4: Second Amplification System

[0056] The PCR premix (PK512) in Tables 3 and 4 contains high-fidelity DNA polymerase, dNTPs, and Mg. 2+ PCR reaction solution containing all necessary components for amplification.

[0057] In this embodiment, the specific steps for amplifying the long fragment of the SLC14A1 gene are as follows: The first amplification system was amplified to obtain the first amplification product; wherein the amplification operating parameters were: 94℃, pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 62℃ annealing for 30 seconds, 68℃ extension for 5 minutes (to adapt to a product length of about 10kb), 30 cycles; 4℃, storage.

[0058] The second amplification system was amplified to obtain the second amplification product. The amplification parameters were as follows: 94℃ for 2 minutes of pre-denaturation; 98℃ for 10 seconds of denaturation; 62℃ for 30 seconds of annealing; 68℃ for 10 minutes of extension (to accommodate a product length of approximately 20 kb); 30 cycles; and storage at 4℃.

[0059] The specific amplification reaction procedure is shown in Table 5-6.

[0060] Table 5: First Amplification Reaction Procedure

[0061] Table 6: Second Amplification Reaction Procedure

[0062] In this embodiment, the steps for mass control of amplification products include: The products of the long-fragment amplification reaction were analyzed using 0.8% agarose gel electrophoresis to determine whether the target gene had been amplified. Voltage: 120V, Time: 40 minutes; Electrophoresis confirmed that the amplified products were of the correct size and that no specific bands (such as...) were present. Figure 2 (As shown).

[0063] The first and second amplification products were mixed in equimolar amounts and used as samples for the subsequent construction of nanopore sequencing libraries.

[0064] Example 3: Construction of Nanopore Sequencing Libraries The nanopore sequencing library construction method of this embodiment, for the long fragment amplification product obtained in Example 2 (i.e., the mixture of the first amplification product and the second amplification product in Example 2), adopts the following steps to construct a library directly adapted for nanopore sequencing: 1) Purification and quantification of long fragment amplification products: The long fragment amplification products of Example 2 were purified using magnetic beads and their concentrations were 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 and sequencing adapters with different tags corresponding to the nanopore sequencer, the adapters are ligated to the end repair products of different samples using ligase; the barcode adapters tag different samples for subsequent data splitting; the sequencing adapters provide chip binding sites + primer binding sites. Without adapters, the sequencing instrument cannot start the sequencing reaction.

[0065] 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.

[0066] The specific steps are explained below: First, purify the long fragment amplification product, as follows: 1) Purification of long-fragment amplification products 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 6) Use the Qubit nucleic acid quantification instrument to determine the concentration of the purified amplified product.

[0072] Then, the purified long fragment amplification product was end-repaired using end-repair enzymes and DNA repair enzymes. The specific steps for end-repair are as follows: 500 ng of purified amplification product was added to a centrifuge tube, and the purified amplification product was end-repaired. The end-repair system is shown in Table 7, and the repair procedure is shown in Table 8.

[0073] Table 7: End-of-Life Remediation Systems

[0074] Table 8: Repair Procedure

[0075] After end repair, barcode adapters with different labels are connected to the end repair product. The specific connection steps are as follows: using the barcode adapter corresponding to the nanopore sequencer, the barcode adapter is connected to the end repair product by ligase.

[0076] The barcode-linked reaction system is shown in Table 9.

[0077] Table 9: Barcode Linked Reaction System

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 3) Add 50 μL of nuclease-free water to resuspend the magnetic beads and let stand at room temperature for 3 minutes.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 9) Use the Qubit nucleic acid quantification instrument to determine the concentration of the purified barcode-linked sample mixed library.

[0088] Next, the sequencing adapter ligation reaction will be performed.

[0089] In this embodiment, the sequencing adapter ligation reaction system is shown in Table 10.

[0090] Table 10: Sequencing adapter ligation reaction system

[0091] 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.

[0092] Finally, the final library was purified using magnetic beads to remove unligated adapters, ligation buffer, and enzymes. The purification method included the following steps: 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 5) After purification, the product concentration is measured using a Qubit nucleic acid quantification instrument, which is the final sequencing library.

[0097] Example 4: Nanopore Sequencing and Data Analysis In this embodiment, the sequencing system configuration and sequencing steps are as follows: 35 fmol of the final library is mixed with the sequencing reaction buffer and loaded onto the sequencing chip of the nanopore sequencer. In this embodiment, the sequencer platform is QiCarbon QPursue-6k.

[0098] like Figure 3 As shown, the sequencer's parameter setting interface allows users to set parameters such as sequencing time, data volume threshold, and quality threshold (quality value > 7) according to specific sequencing needs. The sequencing software is then run, and the nanopore sequencer performs real-time barcode decoding and base identification. This generates raw sequence data in FASTQ format, obtaining long-read sequence data for the SLC14A1 gene.

[0099] In this embodiment, the steps for analyzing sequencing data include: 1) Sequence alignment and variant identification: The sequencing read data is aligned to the SLC14A1 gene reference sequence using analysis software, and variant identification is performed through the alignment.

[0100] 2) Haplotype typing and result interpretation: Linkage was determined directly from read length data covering the c.838 site and other variant sites. Results are shown in [link to results]. Figure 4 , Figure 4 This is an example diagram of haplotype analysis of the Kidd blood group coding gene SLC14A1 using long read data, as shown in Example 4. It illustrates multiple SNP sites covered on the read data.

[0101] 3) Based on the identified haplotype combinations, the final SLC14A1 genotype was determined by referring to the ISBT database. The results are shown in Table 11.

[0102] Table 11 Kidd blood type genotyping results of samples in Example 4

[0103] The results in Table 11 demonstrate that this method successfully identified all known types.

[0104] 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 amplification of the Kidd blood group system based on long-read nanopore sequencing, characterized in that, The primer set consists of a first primer pair and a second primer pair. composition: The first primer pair, designated JK01, has the upstream primer sequence shown in SEQ ID NO: 1 and the downstream primer sequence shown in SEQ ID NO:

2. The second primer pair, designated JK02, has the upstream primer sequence shown in SEQ ID NO: 3 and the downstream primer sequence shown in SEQ ID NO:

4. The first and second primer pairs are used to amplify the full-length sequence of the Kidd blood type coding gene SLC14A1, and the amplification products are used for long-read sequencing.

2. A genotyping and amplification system for the Kidd blood group system based on long-read nanopore sequencing, characterized in that, The amplification system includes the primer pair as described in claim 1, as well as PCR premix and template DNA.

3. A method for amplifying the SLC14A1 gene, characterized in that, The method includes the following steps: 1) Extracting human genomic DNA; 2) Prepare the amplification system as described in claim 2; 3) Run the 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, extension at 68℃ for 5-10 minutes, cycled 30-35 times; finally, store at 4℃.

4. A method for constructing a library of SLC14A1 gene amplification products based on nanopore sequencing, characterized in that, The method described herein, for the long fragment amplification products obtained by the method as described in claim 3, 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 and their concentrations were 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.

5. A method for sequencing the SLC14A1 gene 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 identification is performed to generate raw sequence data in FASTQ format, and long-read sequence data of the SLC14A1 gene is obtained.

6. A method for SLC14A1 genotyping and haplotype analysis, characterized in that, The method, based on the read length data obtained by the sequencing method as described in claim 5, comprises the following steps: 1) Align high-quality read data to the SLC14A1 gene reference sequence; 2) Identify all SNPs and Indel variants, including c.838G>A; 3) Taking advantage of long read lengths, haplotypes can be determined directly based on the linkage relationships of polymorphic sites on read length data, using multiple variant sites covered on a single read length. 4) Based on the known SLC14A1 allele database, determine the alleles, including JKA, JKB 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 Kidd blood typing, prediction of hemolytic transfusion reaction risk, and identification of rare Kidd blood types.

Images