A primer combination, kit, method and application for high-resolution typing of KIR genes based on transcriptome

Abstract

The application provides a primer combination, a kit, a method and application of high-resolution typing of KIR genes based on a transcriptome, and belongs to the technical field of molecular biology and immunogenetics. The primer combination provided by the application comprises a first primer pair with a sequence as shown in SEQ ID NO. 1-2 and a second primer pair with a sequence as shown in SEQ ID NO. 3-4, and based on the primer combination, the application provides a kit and a KIR gene typing method for high-resolution typing of KIR genes based on a transcriptome, which can be used for comprehensive, balanced and high-resolution expression gene typing and transcript analysis on the KIR gene family with high polymorphism and high homology.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and immunogenetics, specifically to a primer combination, kit, method, and application for high-resolution typing of the KIR gene based on the transcriptome. Background Technology

[0002] The killer-cell immunoglobulin-like receptor (KIR) gene family is a highly polymorphic and homologous group of genes located on the long arm of human chromosome 19 (19q13.4) within the leukocyte receptor complex region. The proteins they encode play a central role in the regulation of innate and adaptive immunity. KIR proteins are primarily expressed on the surface of natural killer (NK) cells and some T cells. By recognizing human leukocyte antigen (HLA) class I molecules, they transmit inhibitory or activating signals, thereby precisely regulating the activity of immune cells and maintaining immune homeostasis. Numerous studies have confirmed that specific KIR genotypes, alleles, and even haplotypes are closely related to an individual's susceptibility to various diseases and clinical outcomes, including viral infections (such as HIV, HCV, CMV, and COVID-19), autoimmune diseases, pregnancy complications, and the occurrence and development of malignant tumors. Especially in the field of hematopoietic stem cell transplantation, KIR-ligand incompatibility between donors and recipients is considered a key factor affecting post-transplant relapse, graft-versus-host disease (GVHD), and overall survival. Furthermore, in cancer immunotherapy, KIR genotype is being viewed as a potential biomarker for predicting the efficacy of NK cell therapy and checkpoint inhibitors.

[0003] The complexity of the KIR system manifests on two levels: gene content polymorphism and allelic polymorphism. Different individuals carry KIR genes with significant differences in number and combination, forming diverse haplotypes; simultaneously, each KIR locus (such as KIR2DL1, KIR3DL1, etc.) contains numerous alleles with varying sequences. According to the latest statistics from the IPD-IMGT / KIR international database (Release 2.15, 2025-12), the total number of KIR alleles has reached 2286, with a large number of alleles, proteins, and invalid alleles for each KIR gene. The allele distribution of each KIR gene is extremely uneven. For example, KIR2DL1 and KIR3DL1 are the most polymorphic genes known, with 433 and 319 alleles respectively, posing a significant challenge to high-resolution genotyping. Some activating KIR genes also exhibit significant diversity, such as KIR2DS5 (88 alleles) and KIR3DS1 (92 alleles). Notably, all 42 alleles at the KIR2DS4 locus are classified as null alleles, and their high frequency in the population complicates the interpretation of their function. Although the pseudogenes KIR2DP1 and KIR3DP1 do not encode functional proteins, they still possess 115 and 188 allele sequences respectively, and their presence may influence the evolution and genotyping of functional genes through recombination events. High allele polymorphism and sequence homology exceeding 97% between genes together constitute the core of the complexity of the KIR system.

[0004] At the structural level of KIR genes, there are key differences in composition between their mRNA transcripts and full-length genomic DNA sequences: mRNA contains only exon sequences (including coding regions and 5' and 3' untranslated regions, UTRs), without introns; while genomic DNA contains exons, introns, and 5' and 3' UTR sequences. Furthermore, the 5' and 3' UTR regions of different members of the KIR gene family show significant differences in length and sequence composition between genomic DNA and mRNA, and the trends in UTR length variation are not consistent across different genes. For example, the 5' and 3' UTR sequences of KIR2DL4 show low homology with other KIR genes (such as KIR2DL1, KIR3DL1, etc.) and exhibit significant length differences. These structural differences in UTR regions are crucial for designing primers that can specifically and efficiently amplify the full-length transcripts of different KIR genes. Primer design strategies targeting mRNA templates must fully consider the composition and length variations of the UTR sequences of each gene; otherwise, it is easy to cause problems such as uneven primer binding efficiency, off-target amplification, or even complete failure to obtain the target product.

[0005] In recent years, with the deepening of transcriptomics research, our understanding of KIR gene expression patterns has been continuously expanding. For example, the expression levels of KIR3DL3, KIR2DP1, and KIR3DP1 in whole blood are relatively low (mean TPM of 3.42, 68.79, and 8.63, respectively). Although KIR3DL3 has a complete coding frame, its expression is mainly limited to tissue-resident T cells and specific subsets of NK cells, with low abundance in peripheral circulating immune cells, resulting in a weak signal detected in the whole blood transcriptome. KIR2DP1 and KIR3DP1 are pseudogenes; although they exist intact at the genomic DNA level, their promoter activity is weak, their mRNA stability is low, and they lack complete open reading frames, leading to significantly lower steady-state transcription levels than functional KIR genes. However, these three genes still exhibit considerable expression in specific tissues (e.g., KIR3DL3 in the spleen and testes, and KIR2DP1 and KIR3DP1 in the spleen), suggesting that they may play a regulatory role in the local tissue microenvironment. Therefore, there is an urgent need in this field for a comprehensive KIR detection method that can capture these low-expression genes. At the same time, an ideal detection method should be able to distinguish between "genes present in the genome" and "truly expressed transcripts," thereby providing more accurate information for functional studies and clinical decision-making.

[0006] Currently, some PCR-based methods using sequence-specific primers (PCR-SSP) and sequence-specific oligonucleotide probes (PCR-SSO) exist. While these techniques are simple and rapid, their resolution is limited, typically only allowing for "deletion / presence" genotyping at the gene level. They cannot distinguish fine allelic differences, let alone discover new alleles, severely restricting a deeper understanding of KIR functional diversity. Subsequent developments in Sanger sequencing-based genotyping (PCR-SBT) improved resolution to the allele level. However, due to the extremely high sequence similarity (over 97%) in the coding regions of KIR gene family members (e.g., KIR2DL1 vs. KIR2DL2 / 3, KIR3DL1 vs. KIR3DS1), cross-reactivity and missed allele detection are prone to occur during PCR amplification. More importantly, Sanger sequencing cannot resolve the phase problem, i.e., it is difficult to determine whether polymorphic sites scattered across different gene locations are located on the same chromosome, thus making it impossible to accurately construct haplotypes, especially prominent in heterozygous individuals. The inherent short read length (typically 150-300 bp) of NGS technology necessitates randomly fragmenting DNA samples into small segments to obtain the full-length KIR gene sequence, followed by complex bioinformatics algorithms for assembly. However, in highly homologous regions of KIR genes, this short-read assembly process is highly susceptible to chimeric sequences or misassembly, leading to unreliable genotyping results. Furthermore, for cDNA analysis, short reads struggle to unambiguously cover the entire transcript and accurately reconstruct different alternative splice isoforms. For instance, existing technologies offer a full-length third-generation sequencing genotyping method for KIR genes based on genomic DNA (see patent CN120118992A). This method overcomes the phase problem of Sanger sequencing through long-read sequencing technology, enabling full-length allelic genotyping of 16 KIR genes and significantly improving resolution. However, genomic DNA-based sequencing methods still cannot distinguish between functional genes, pseudogenes (such as KIR2DP1 and KIR3DP1), and null alleles. These sequences, which exist intact at the genomic DNA level, are not necessarily transcribed into functional proteins. Moreover, full-length genomic DNA sequencing requires amplification of long fragments (typically >10 kb) containing complete intron regions, placing extremely high demands on template DNA quality and the robustness of the long fragment amplification system, making the operation complex and costly. Transcriptome sequencing (RNA-Seq), by directly detecting the complementary DNA (cDNA) generated by reverse transcription in a sample, can accurately reflect the expression status of KIR genes, effectively avoiding interference from pseudogenes and non-expressing alleles, thus providing more accurate information for functional studies and clinical decision-making.However, when applying transcriptome sequencing technology to high-resolution genotyping of KIR genes, especially when combined with high-precision long-read sequencing platforms such as PacBio HiFi, a series of new and not yet fully resolved technical challenges arise. (1) Primer design challenges: The cDNA sequences of the KIR gene family are highly similar, and there are a large number of single nucleotide polymorphisms (SNPs) and insertions / deletions (Indels) among different alleles. Designing primer pairs that can amplify the full-length cDNA of all functional KIR genes in a balanced, efficient, and specific manner, without being affected by these sequence variations, is a great challenge. In particular, the 5' and 3' UTR regions of different KIR genes have significant differences in length and sequence composition, which puts forward contradictory requirements for primer binding positions and annealing temperatures. A single "universal" primer pair often fails to cover all important KIR genes, or there are significant deviations in amplification efficiency between different loci, resulting in insufficient sequencing coverage or even complete missed detection of some key alleles (such as KIR2DL4, KIR2DS4, etc.), which cannot truly reflect the transcriptome status of the sample.

[0007] (2) Library construction optimization: The process of constructing high-quality sequencing libraries from cDNA amplicon, which is usually very precious, requires fine optimization. How to maintain the integrity of long fragment amplification products and effectively remove primer dimers, adapter self-ligation products and non-specific amplification products to obtain high-purity sequencing libraries is a prerequisite for ensuring the quality of sequencing data.

[0008] (3) Lack of bioinformatics analysis workflow: Currently, there is a lack of standardized bioinformatics analysis workflows specifically for KIR cDNA third-generation sequencing data. Commercial software or general workflows often cannot effectively handle the high homology and polymorphism unique to the KIR family. Accurately performing sequence deconvolution from mixed sequencing data that may contain multiple highly similar alleles, and precisely clustering reads to their respective genes and alleles, is the key to successful genotyping and is also the biggest technical bottleneck at present.

[0009] Therefore, there is an urgent need in this field for a high-resolution typing method for KIR genes based on transcriptomics, in order to simultaneously achieve high coverage of the KIR gene family, high resolution at the allele level, and a true reflection of expression status. Summary of the Invention

[0010] To address the problems existing in the prior art, the first objective of this invention is to provide a primer combination for high-resolution typing of the KIR gene based on the transcriptome.

[0011] A second objective of this invention is to provide the application of the aforementioned primer combination in high-resolution genotyping of the KIR gene.

[0012] A third objective of this invention is to provide a transcriptome-based KIR genotyping method.

[0013] A fourth objective of this invention is to provide a kit for high-resolution typing of the KIR gene.

[0014] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer combination for high-resolution typing of the KIR gene based on transcriptome, the primer combination comprising a first primer pair and a second primer pair; the upstream primer sequence of the first primer pair is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2; the upstream primer sequence of the second primer pair is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4.

[0015] Preferably, the first primer pair is used to amplify other KIR genes besides the KIR2DL4 gene, including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1, and KIR2DP1; the second primer pair is used to amplify the KIR2DL4 gene.

[0016] This invention also provides the application of the above primer combinations in high-resolution typing of the KIR gene.

[0017] This invention also provides a transcriptome-based KIR gene typing method for non-disease diagnosis or treatment purposes, comprising the following steps: using sample RNA as a template, performing reverse transcription PCR amplification using the first primer pair and the second primer pair from the above primer combination; mixing the first amplification product obtained by amplification using the first primer pair with the second amplification product obtained by amplification using the second primer pair at a mass ratio of 30:2 to obtain a mixed sequencing library; sequencing the mixed sequencing library to obtain the sequence information of the KIR gene.

[0018] Preferably, the reverse transcription PCR amplification system includes: 12.5 μl of 2×FastKing One Step RT-PCR MasterMix, 1 μl of 25×RT-PCR Enzyme Mix, 0.8 μl of 10 μM upstream primer, 0.8 μl of 10 μM downstream primer, 5 μl of 40~100 ng / μL RNA template, and 4.9 μl of RNase-Free ddH2O.

[0019] Preferably, the amplification conditions for the first primer pair include: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 65℃ for 30 sec, and 72℃ for 30 sec; and extension at 72℃ for 5 min.

[0020] Preferably, the amplification conditions for the second primer pair include: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 59℃ for 30 sec, and 72℃ for 30 sec; and extension at 72℃ for 5 min.

[0021] The present invention also provides a kit for high-resolution typing of the KIR gene, the kit comprising the above-described primer combination.

[0022] Preferably, the kit also includes reagents for reverse transcription PCR reactions.

[0023] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention systematically solves the problem of KIR gene amplification bias through a pioneering "separate amplification + optimized mixing" strategy. It is the first to recognize the inherent difference in amplification efficiency between KIR2DL4 and other KIR genes, and creatively employs two independent primer pairs for separate amplification, fundamentally avoiding the missed detection of key genes due to uneven amplification efficiency caused by a single primer pair. This strategy is completely different from the existing approach that attempts to balance amplification efficiency by designing "universal primers" or adjusting the number of cycles.

[0024] This invention overcomes the bottleneck of low amplification efficiency of the KIR2DL4 gene by employing a "pooled amplification + optimized mixing" strategy. It is the first to achieve balanced detection of the KIR gene family (especially the low-amplification-efficiency KIR2DL4). Furthermore, based on transcriptome analysis, it can directly reflect the true KIR allele composition expressed in the sample, avoid pseudogene interference, and prevent gene omissions and read bias caused by single primer amplification. The operation is simple, cost-controllable, and highly compatible with third-generation sequencing technology. It can provide reliable technical support for high-resolution KIR gene typing, hematopoietic stem cell transplantation donor selection, and leukemia immunotherapy evaluation. Attached Figure Description

[0025] Figure 1 : Schematic diagram of the overall technical route of the method of the present invention.

[0026] Figure 2The image shows electrophoresis images of amplification products from representative samples using the first and second primer pairs of this invention. The left image is a UV-Vis gel electrophoresis image of the amplification products from a KIR AA1 genotype sample (according to the AFND database standard number), which carries genes 2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2, 3DL3, 2DP1, and 3DP1. The right image is a UV-Vis gel electrophoresis image of the amplification products from a KIR AB2 genotype sample (according to the AFND database standard number), which carries genes 2DL1, 2DL3, 2DL4, 2DL5, 2DS1, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. The first well for each genotype is a marker (DL2000 DNA molecular weight standard, bands from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100...). The first well contains the amplification product of the first primer pair, and the second well contains the amplification product of the second primer pair.

[0027] Figure 3 Visualization of the alignment of KIR transcriptome sequences of individuals with the KIR AA1 genotype (according to the AFND database standard number) obtained by the method of this invention with the reference genome.

[0028] Figure 4 Visualization of the alignment of KIR transcriptome sequences of individuals with the KIR BB75 genotype (according to the AFND database standard number) obtained by the method of this invention with the reference genome.

[0029] Figure 5 Electrophoresis images of amplification results for six primer pairs targeting KIR genes other than KIR2DL4; lane 1 shows the marker (DL2000 DNA molecular weight standard, bands from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); lanes 2-5 show the amplification products of sample 006626 (KIR genotype AB5) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃); lanes 6-9 show the amplification products of sample 006627 (KIR genotype AA1) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃).

[0030] Figure 6Electrophoresis images of amplification products from two primer pairs targeting KIR2DL4; lane 1 shows the marker (DL2000 DNA molecular weight standard, bands from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); lanes 2-5 show the amplification products of sample 006626 (KIR genotype AB5) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃); lanes 6-9 show the amplification products of sample 006627 (KIR genotype AA1) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃). Detailed Implementation

[0031] In the course of long-term research on KIR genotyping technology, this invention has found that the core reason why a single primer pair in the existing technology cannot evenly cover all KIR genes is that the sequence characteristics of the KIR2DL4 gene are significantly different from those of other KIR genes (such as KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, etc.). When using the same primer pair for amplification, the amplification efficiency of KIR2DL4 is much lower than that of other KIR genes, causing it to be submerged in mixed sequencing.

[0032] However, designing primer combinations capable of specifically and uniformly amplifying the full-length transcripts of the KIR gene family is technically far more challenging than conventional PCR primer design. First, the coding region sequences among members of the KIR gene family exhibit extremely high homology (up to 97%), making primers recommended by conventional primer design software (such as Primer-BLAST) prone to cross-amplification among multiple genes. Second, the 5'UTR and 3'UTR of different KIR genes differ significantly in length and sequence composition: if primers are anchored to relatively conserved UTR regions to cover the complete coding region, sequence variations in these regions will make a single primer incompatible with all alleles; if primers are designed only for the coding region, it becomes difficult to distinguish highly homologous family members. Furthermore, mRNA does not contain introns, and its sequence composition differs from the corresponding genomic DNA. Primers designed based on genomic DNA sequences often fail in cDNA amplification because their binding sites are located in intronic regions or across exon-intron boundaries. The interplay of these multiple factors necessitates that the primer design of this invention simultaneously consider cross-gene specificity, cross-allelic inclusiveness, and adaptability to the mRNA template, which cannot be addressed using conventional strategies such as "selecting conserved regions" or "degenerate base design." This invention ultimately designs and screens specific primer sequence combinations (SEQ ID NO.1~SEQ ID NO.4) that simultaneously meet the aforementioned stringent requirements.

[0033] This invention creatively proposes a strategy of "pooled amplification and optimized mixing" (see schematic diagram of the overall technical route). Figure 1 As shown in the figure): KIR2DL4 was amplified separately from other KIR genes, and the optimal mixing ratio of the two amplification products was proposed so that all KIR genes could obtain balanced read coverage in one sequencing reaction.

[0034] This invention provides a primer pair for high-resolution typing of the KIR gene based on transcriptome, the primer pair comprising a first primer pair and a second primer pair; the upstream primer sequence (KIR-RNA-TGS-F) of the first primer pair is 5'-CCGGCASCACCATGTCRCTC-3', as shown in SEQ ID NO.1, and the downstream primer sequence (KIR-RNA-TGS-R) is 5'-AGAGGTGGGCAGGGGTCAAGT-3', as shown in SEQ ID NO.2; the upstream primer sequence (KIR2DL4-RNA-TGS-F) of the second primer pair is 5'-AATGTGTCAACTGCACGATCCGG-3', as shown in SEQ ID NO.3, and the downstream primer sequence (KIR2DL4-RNA-TGS-R) is 5'-GGAAGAGTGATGCTCTAAGATGG-3', as shown in SEQ ID NO.4.

[0035] In this invention, the first primer pair is used to amplify other KIR genes besides the KIR2DL4 gene, including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1, and KIR2DP1 (excluding KIR3DP1, as the downstream primer cannot bind); the amplification product covers the core coding region of the KIR gene (covering the main polymorphic sites), with an expected length of approximately 1.2~1.6kb; the second primer pair is used to amplify the KIR2DL4 gene; the amplification product covers the full-length coding region of KIR2DL4, with an expected length of approximately 1.3kb. The binding positions of the first primer pair and the second primer pair on their respective targeted KIR genes and the lengths of the amplification products are shown in the table below (based on the alignment results of representative allele reference sequences of each gene in the IPD-IMGT / KIR database Release 2.15).

[0036] Table 1. Binding sites and amplification product lengths of the first and second primer pairs on their respective target KIR genes.

[0037] The two primer pairs designed in this invention have been experimentally verified. The first primer pair (KIR-RNA-TGS-F / KIR-RNA-TGS-R) exhibits diffuse or diffuse-band mixed bands in the 1.2~1.6kb range, successfully capturing all functional KIR genes and pseudogene transcripts except KIR2DL4. The second primer pair (KIR2DL4-RNA-TGS-F / KIR2DL4-RNA-TGS-R) presents a clear single target band at approximately 1.3kb, specifically amplifying the full-length coding region of KIR2DL4. The primers of this invention are precisely designed, have high amplification specificity, and can effectively distinguish KIR2DL4 from other highly homologous KIR genes.

[0038] This invention also provides the application of the above primer combinations in high-resolution KIR gene genotyping. Based on the two primer pairs designed in this invention, the reaction system and amplification conditions were further optimized, forming a complete transcriptome-based KIR gene detection protocol.

[0039] The transcriptome-based KIR gene typing method provided by this invention includes the following steps: using sample RNA as a template, reverse transcription PCR amplification is performed using the first primer pair and the second primer pair in the above primer combination, respectively; the first amplification product obtained by amplification using the first primer pair and the second amplification product obtained by amplification using the second primer pair are mixed at a mass ratio of 30:2 to obtain a mixed sequencing library; the mixed sequencing library is sequenced to obtain the sequence information of the KIR gene.

[0040] This invention provides a specific mixing ratio of amplification products (30:2), which ensures that the sequencing read percentage of KIR2DL4 remains stably within the ideal range of 5-10%, while guaranteeing sufficient read coverage for other KIR genes, resulting in optimal performance in terms of signal balance and data utilization. Comparative experiments using different mixing ratios show that: with an equal mixing ratio (1:1), the KIR2DL4 read percentage reaches as high as 42.3%, severely crowding out sequencing resources for other KIR genes; at ratios of 30:5 and 20:5, the KIR2DL4 read percentages are 15.6% and 12.8%, respectively, and in some low-expression samples, they can further increase to over 20%, affecting library balance; while at a 30:2 ratio, the average KIR2DL4 read percentage is 8.2% ± 1.3%, consistently maintained within the ideal range of 5-10%, while ensuring sufficient read coverage for other KIR genes, resulting in the highest total number of effective reads (average 92,156). This invention achieves precise balance of sequencing reads by controlling the mixing ratio. The KIR2DL4 gene with low amplification efficiency can obtain sufficient sequencing depth to support high-resolution genotyping, while highly expressed or other efficiently amplified genes do not excessively occupy sequencing resources, and it performs best in terms of signal balance and data utilization.

[0041] Preferably, the reverse transcription PCR amplification system (25 μl) of the present invention comprises: 12.5 μl of 2×FastKing One StepRT-PCR Master Mix, 1 μl of 25×RT-PCR Enzyme Mix, 0.8 μl of 10 μM upstream primer, 0.8 μl of 10 μM downstream primer, 5 μl of 40~100 ng / μL RNA template, and 4.9 μl of RNase-Free ddH2O. The upstream and downstream primers in this system correspond to the upstream and downstream primers of the first primer pair, or the upstream and downstream primers of the second primer pair, respectively.

[0042] Preferably, the amplification conditions for the first primer pair of the present invention include: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 65℃ for 30 sec, and 72℃ for 30 sec; and extension at 72℃ for 5 min. The amplification conditions for the second primer pair include: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 59℃ for 30 sec, and 72℃ for 30 sec; and extension at 72℃ for 5 min.

[0043] The hybrid sequencing library provided by this invention enables downstream bioinformatics analysis to perform accurate sequence deconvolution and genotyping based on sufficient read coverage, avoiding analysis failures or unreliable results due to insufficient reads of certain genes, and better reflecting the true allelic composition of expression.

[0044] Preferably, when sequencing the mixed sequencing library, the present invention employs third-generation sequencing technology, including but not limited to PacBio single-molecule real-time sequencing (SMRT) or Oxford Nanopore nanopore sequencing. More preferably, the present invention employs PacBio high-fidelity (HiFi) sequencing mode to obtain the sequence information of the KIR gene.

[0045] The hybrid sequencing library described in this invention is perfectly compatible with third-generation sequencing technology, and is particularly suitable for use with third-generation sequencing platforms such as PacBio HiFi. Long-read sequencing technology can directly span KIR gene transcripts. Combined with the hybrid sequencing library obtained by this invention, complete, splice-free, and highly accurate full-length sequences of all KIR genes can be obtained in one go, while simultaneously solving the splicing error problem of short-read NGS and the incomplete coverage problem of single-primer amplification. Actual sequencing data show that the average length of HiFi reads in each sample is between 1406 and 1515 bp, and the average read quality is ≥Q20 (accuracy ≥99%), which can be directly used for KIR gene full-length sequence alignment and high-resolution genotyping.

[0046] The transcriptome-based KIR genotyping method provided by this invention is simple to operate, cost-effective, and easy to standardize. It requires only two independent RT-PCR reactions and a simple mixing step, eliminating the need for complex nested PCR, haplotype isolation, or multiple rounds of amplification, significantly reducing operational complexity and reagent costs. The standardized system and conditions make it easy to transfer and reproduce between different laboratories, making it suitable for widespread application in clinical testing services.

[0047] The transcriptome-based KIR gene typing method provided by this invention has unique advantages based on transcriptome: it reflects true expression and avoids pseudogene interference. This invention detects the KIR transcripts that are actually expressed in the sample, rather than the static gene sequences that exist in the genome. This characteristic enables it to: (1) avoid pseudogene interference: pseudogenes such as KIR2DP1 and KIR3DP1 exist at the gDNA level but are usually not translated into functional proteins. This invention, through primer design and transcriptome-based analysis, can avoid misjudging their signals as functional genes, or fundamentally eliminate the invalid amplification and interference of pseudogenes; (2) discover allele expression imbalance: it can identify differential expression of alleles in heterozygotes, providing key clues for functional studies; (3) directly reflect immune status: the expression level of KIR genes is closely related to NK cell activation status, disease progression and treatment response. This invention can obtain typing and relative expression information simultaneously, which is superior to simple gDNA typing methods.

[0048] This invention also provides a kit for high-resolution genotyping of the KIR gene, the kit comprising the primer combination described above. Preferably, the kit further comprises reagents for reverse transcription PCR, including 2×FastKingOne Step RT-PCR Master Mix, 25×RT-PCR Enzyme Mix, and RNase-Free ddH2O. Alternatively, the kit may further comprise purification magnetic beads or purification columns for purifying the amplification products.

[0049] The primer combinations, kit methods, or KIR genotyping methods described above in this invention are not only applicable to peripheral blood samples, but can also be extended to RNA samples from various tissues such as the spleen, lungs, intestinal mucosa, and salivary glands. Furthermore, the ALL risk genes KIR2DL1 (OR=2.4) and KIR3DL1 (OR=10.87), as well as the protective gene KIR2DS4 (OR=0.15), which are highly correlated with leukemia genetic association studies, are the core detection targets of the methods of this invention.

[0050] The primer combinations or kits provided by this invention can be used for the actual development of the following functions: (1) screening hematopoietic stem cell transplant donors; (2) evaluating the efficacy of NK cell immunotherapy; (3) precision diagnosis and treatment of leukemia; and (4) discovering and identifying new allele variants.

[0051] This method can provide direct technical support for the selection of hematopoietic stem cell transplant donors, the evaluation of the efficacy of NK cell immunotherapy, and the precision diagnosis and treatment of leukemia. It has broad tissue applicability and clinical translation potential.

[0052] The transcriptome sequencing-based strategy of this invention can directly capture the actual expressed transcript sequences, avoiding missed detections caused by primer mismatch or allele dropout. It is particularly beneficial for discovering low-frequency or population-specific new variants, and shows significant advantages in detection accuracy, balance, and ability to discover new variants. It has good prospects for scientific research and clinical translation.

[0053] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] Unless otherwise specified, the following embodiments are all conventional methods.

[0055] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0056] Example 1 Primer design and synthesis for high-resolution KIR gene typing: Based on the full-length cDNA sequences of each member of the KIR gene family in the NCBI database, multiple sequence alignment was performed using ClustalW to screen for regions that are highly conserved in other KIR genes except KIR2DL4, but specifically present in KIR2DL4. Two pairs of primers were then designed for each region. First primer pair (used to amplify other KIR genes: including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1 and KIR2DP1, excluding KIR3DP1): The upstream primer sequence (KIR-RNA-TGS-F) is 5'-CCGGCASCACCATGTCRCTC-3', as shown in SEQ ID NO.1; The downstream primer sequence (KIR-RNA-TGS-R) is 5'-AGAGGTGGGCAGGGGTCAAGT-3', as shown in SEQ ID NO.2; Expected amplification product length: approximately 1.2~1.6 kb (covering the core coding region of the KIR gene).

[0057] Second primer pair (for specific amplification of KIR2DL4): The upstream primer sequence (KIR2DL4-RNA-TGS-F) is 5'-AATGTGTCAACTGCACGATCCGG-3', as shown in SEQ ID NO.3; The downstream primer sequence (KIR2DL4-RNA-TGS-R) is 5'-GGAAGAGTGATGCTCTAAGATGG-3', as shown in SEQ ID NO.4; Expected amplification product length: approximately 1.3 kb (covering the full-length coding region of KIR2DL4).

[0058] All primers were synthesized by Hunan Aikerui Biotechnology Co., Ltd., and purified by HPLC with a purity of ≥95%.

[0059] Example 2 A transcriptome-based KIR genotyping method: (1) Using the sample RNA as a template, reverse transcription PCR amplification was performed using the first primer pair and the second primer pair from Example 1, respectively; two reaction systems were prepared, each system consisting of the following (25 μl system): 2×FastKing One Step RT-PCR Master Mix 12.5μl, 25×RT-PCR Enzyme Mix 1μl, upstream primer 10μM 0.8μl, downstream primer 10μM 0.8μl, RNA template 40~100 ng / μL 5μl, RNase-FreeddH2O 4.9μl.

[0060] Amplification conditions for the first primer pair: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 65℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0061] Amplification conditions for the second primer pair: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 59℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0062] (2) The first amplification product obtained by amplification using the first primer pair and the second amplification product obtained by amplification using the second primer pair were mixed at a mass ratio of 30:2 to construct a sequencing library: If using the PacBio platform for sequencing, prepare the SMRTbell library as follows: purify the mixed product using AMPure PB magnetic beads (PacBio) to remove primer dimers and small molecule impurities; perform end repair and adapter ligation according to the PacBio SMRTbellTemplate Prep Kit 1.0 instructions; digest unligated adapters and primers using Exonuclease III and Exonuclease VII; perform fragment selection using the BluePippin system (Sage Science) to enrich target fragments of 1.0–1.6 kb; and quantify and quality control the library using Qubit and Agilent 2100 Bioanalyzer.

[0063] If using the ONT platform for sequencing, prepare the library using the ONT Ligation Sequencing Kit according to the instructions.

[0064] (3) Sequencing the constructed sequencing library to obtain the sequence information of the KIR gene.

[0065] If using PacBio sequencing: Anneal the SMRTbell library, bind the sequencing primers, and then bind them to DNA polymerase before sequencing. Use the PacBio Sequel IIe or Revio system in CCS (Circular Consensus Sequencing) mode for sequencing, setting the acquisition time to 24 hours. After sequencing, use SMRT Link software (v11.0) to extract HiFi reads, requiring HiFi read accuracy ≥ Q20 (99%).

[0066] If using ONT sequencing: Load the library into an R9.4.1 or R10.4 flow cell and perform sequencing on a MinION or PromethION platform, setting the sequencing time to 48–72 hours. Use Guppy software to identify bases and extract reads from the sequenced data.

[0067] (4) Perform bioinformatics analysis on the obtained KIR gene sequence information.

[0068] Read filtering: Perform quality filtering on off-machine reads, and remove reads with a length <500 bp or a quality value <Q10.

[0069] Reference sequence alignment: Using each KIR gene reference sequence in the IPD-IMGT / KIR database (Release 2.14) as an index, use minimap2 (parameter: -ax map-pb) or BLASTN (E-value ≤ 1e -10 ) for read alignment.

[0070] Genotyping: Use a KIR genotyping-specific algorithm (such as KIR IMP, TYPEr or a self-developed clustering algorithm based on maximum likelihood estimation) to perform allele-level genotyping on each read. For each KIR locus, alleles with the number of reads ≥3 and consistency ≥99% are screened out as positive genotyping results.

[0071] Result output: Output the KIR gene presence / absence status, allele genotyping results (4-digit resolution) and relative expression level of each gene (based on read count) of each sample.

[0072] Alignment result visualization: After aligning the KIR reads of each sample to the reference genome, use IGV (Integrative Genomics Viewer) for visual display.

[0073] Example 3 A kit for high-resolution genotyping of KIR genes, comprising: (1) Primer mixture: The first primer pair (KIR-RNA-TGS-F, KIR-RNA-TGS-R, 10 μM each) described in Example 1 is in a separate tube; the second primer pair (KIR2DL4-RNA-TGS-F, KIR2DL4-RNA-TGS-R, 10 μM each) described in Example 1 is in a separate tube; (2) Reaction premix: 2×FastKing One Step RT-PCR Master Mix (1 tube, 600 μl), 25×RT-PCR Enzyme Mix (1 tube, 50 μl); (3) RNase-Free ddH2O (1 tube, 1 ml); All components of the kit are stored at -20°C, and repeated freezing and thawing are avoided.

[0074] Test Example 1 Transcriptome-based KIR gene genotyping was performed on peripheral blood samples (12 samples).

[0075] 1. Sample processing and RNA extraction Two ml of EDTA-anticoagulated peripheral blood was collected, and total RNA was extracted using the Trizol method (Invitrogen). The procedure is as follows: (1) After mixing peripheral blood with an equal volume of PBS, slowly add it to a centrifuge tube containing 3 ml of Trizol, mix thoroughly, and let stand at room temperature for 5 minutes; (2) Add 0.2 times the volume of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and then centrifuge at 4℃ and 12000×g for 15 minutes; (3) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 minutes. (4) Centrifuge at 4℃ and 12000×g for 10 minutes, discard the supernatant, and wash the precipitate twice with 75% ethanol; (5) After air drying, dissolve the RNA precipitate with 20 μl RNase-Free ddH2O; (6) Use DNase I (Thermo Fisher) to remove residual genomic DNA; (7) Determine RNA concentration and purity, requiring A 260 / A 280 The ratio is between 1.9 and 2.1, A 260 / A 230 The ratio is ≥1.8.

[0076] RNA samples should be stored at -80℃ for future use.

[0077] 2. Reverse transcription PCR amplification was performed using the first primer pair and the second primer pair from Example 1, respectively. Using RNA samples as templates, two reaction systems were prepared, each with the following composition (25 μl): 2×FastKing One Step RT-PCR Master Mix 12.5μl, 25×RT-PCR Enzyme Mix 1μl, upstream primer 10μM 0.8μl, downstream primer 10μM 0.8μl, RNA template 40~100 ng / μL 5μl, RNase-FreeddH2O 4.9μl.

[0078] Amplification conditions for system A (first primer pair): reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 65℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0079] Amplification conditions for system B (second primer pair): reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles at 94℃ for 30 sec, 59℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0080] 3. Product detection and quantification (1) Take 5 μl of the amplification product from each system and perform electrophoresis on a 1.5% agarose gel (containing 0.5 μg / ml GoldView nucleic acid dye). Electrophoresis conditions: 120V, 30 minutes. Observe and photograph using a gel imaging system. The results are as follows: Figure 2 As shown.

[0081] The left image shows the UV-Vis gel electrophoresis image of the amplification products of the KIR AA1 genotype sample, which carries the genes 2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2, 3DL3, 2DP1, and 3DP1. The right image shows the UV-Vis gel electrophoresis image of the amplification products of the KIR AB2 genotype sample, which carries the genes 2DL1, 2DL3, 2DL4, 2DL5, 2DS1, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. The first well for each genotype is a marker (DL2000 DNA molecular weight standard, bands from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp), the second well is the amplification product of the first primer pair, and the third well is the amplification product of the second primer pair.

[0082] The results showed that the amplification products of system A exhibited diffuse or diffuse-band mixed bands within the expected size range (1.2~1.6kb), consistent with the characteristics of simultaneous amplification of multiple KIR genes; the amplification products of system B showed a clear and single target band at approximately 1.3kb.

[0083] (2) The amplification products from both tubes were accurately quantified using a Qubit 4.0 fluorescence quantitative analyzer (Thermo Fisher) and a dsDNA HS Assay Kit. The amplification products from 12 samples were quantified using Qubit, and the results are shown in the table below: Table 2. Detection results of concentration, total amount, and purity of the amplification products of the first and second primer pairs.

[0084] The results showed that the concentration of the amplification product from the first primer pair ranged from 61.6 to 113.2 ng / μl, and the concentration of the amplification product from the second primer pair ranged from 0.44 to 28.2 ng / μl. The concentrations of products in system A (denoted as C_A) and system B (denoted as C_B) were recorded. These data indicate that the amplification efficiency of KIR2DL4 is significantly lower than that of other KIR genes, with its amplification product concentration averaging only 1 / 10 to 1 / 50 of that of the first primer pair. Direct mixing would result in an excessively low proportion of KIR2DL4 in the sequencing data, making effective genotyping impossible. This demonstrates the advantage of this invention in achieving sequencing read balance by adjusting the mixing ratio (30:2).

[0085] 4. Product mixing and sequencing library preparation (1) Based on the Qubit quantification results, calculate the required volume using the following formula to aspirate amplification products equivalent to 30 ng and 2 ng respectively: V_A = (30 / C_A) μl (round to the integer, corresponding to a mass of 30ng); V_B = (2 / C_B) μl (round to the integer, corresponding to a mass of 2ng); (2) The calculated volumes of products A and B were mixed in a 1.5 ml low-adsorption centrifuge tube, and the volume was brought up to 20 μl using RNase-Free ddH2O. After mixing, the mixture was briefly centrifuged. The product concentrations after mixing of the 12 samples were 85.6 ng / μl, 85.6 ng / μl, 70.8 ng / μl, 92.8 ng / μl, 77.6 ng / μl, 112.4 ng / μl, 64.0 ng / μl, 79.6 ng / μl, 76.0 ng / μl, 79.2 ng / μl, 62.0 ng / μl, and 60.4 ng / μl, respectively, and the total amounts all met the requirements for library preparation.

[0086] (3) Constructing sequencing libraries: If using the PacBio platform for sequencing, prepare the SMRTbell library as follows: purify the mixed product using AMPure PB magnetic beads (PacBio) to remove primer dimers and small molecule impurities; perform end repair and adapter ligation according to the PacBio SMRTbellTemplate Prep Kit 1.0 instructions; digest unligated adapters and primers using Exonuclease III and Exonuclease VII; perform fragment selection using the BluePippin system (Sage Science) to enrich target fragments of 1.0–1.6 kb; and quantify and quality control the library using Qubit and Agilent 2100 Bioanalyzer.

[0087] If sequencing is performed using the ONT platform, library preparation is carried out using the ONT Ligation Sequencing Kit according to the manufacturer's instructions.

[0088] 5. On-machine sequencing: PacBio sequencing: The SMRTbell library is annealed and bound to sequencing primers, then bound to DNA polymerase before being loaded onto the sequencer. Sequencing is performed using the PacBio Sequel IIe or Revio system in CCS (Circular Consensus Sequencing) mode, with the acquisition time set to 24 hours. The off-machine data is processed with SMRT Link software (v11.0) for HiFi read extraction, requiring HiFi read accuracy ≥ Q20 (99%).

[0089] Table 3 Statistics of HiFi sequencing data for each sample

[0090] The actual number of HiFi reads obtained for each sample through sequencing ranges from 4709 to 21457, the average read length ranges from 1406 to 1514 bp, and the average read quality ranges from 0.998 to 0.999 (i.e., accuracy ≥ 99.8%).

[0091] 6. Bioinformatics analysis (1) Read filtering: Quality filtering is performed on the off-machine reads to remove reads with length <500 bp or quality value <Q10.

[0092] (2) Reference sequence alignment: Using the reference sequences of each KIR gene in the IPD-IMGT / KIR database (Release 2.14) as the index, minimap2 (parameter: -ax map-pb) or BLASTN (E-value ≤ 1e -10 ) is used for read alignment.

[0093] (3) Genotyping: A KIR genotyping-specific algorithm (such as KIR IMP, TYPEr or a self-developed clustering algorithm based on maximum likelihood estimation) is used to perform allele-level genotyping on each read. For each KIR locus, alleles with a read count ≥3 and a consistency ≥99% are screened as positive genotyping results.

[0094] (4) Results output: Output the presence / deletion status of KIR genes, allele typing results (4-digit resolution), and relative expression levels of each gene (based on read count) for each sample. The statistical results of the 12 samples based on the pooled transcriptome analysis showed that the reads of each KIR gene locus were evenly distributed, and the number of KIR2DL4 reads was between 68 and 311, maintaining an ideal proportion of 5% to 15%.

[0095] (5) Visualization of alignment results: After aligning the KIR reads of each sample to the reference genome, the results are visualized using IGV (Integrative Genomics Viewer). Figure 3 and Figure 4 As shown.

[0096] Figure 3 This shows the alignment of the KIR transcriptome sequences of individuals with the KIR AA1 genotype with the reference genome. Figure 4 The comparison results of individuals with the KIR BB75 genotype are shown. The figures clearly demonstrate that the read coverage of each KIR locus (including KIR2DL4) is uniform, with no obvious gaps or biases, further validating the effectiveness of the method of this invention in the balanced detection of KIR gene families.

[0097] Experimental Example 2 Comparative experiments with different mixing ratios: To verify the superiority of the 30:2 mixing ratio, a comparative experiment was set up with different mixing ratios: following the method in Experiment Example 1, the amplification products of system A and system B of the same sample were taken and mixed at mass ratios A:B = 1:1, 30:2, 30:5, and 20:5, respectively. Three replicates were set up for each ratio. Then, library preparation and PacBio sequencing were performed in the same manner as in Experiment Example 1 (sequencing in the same batch to eliminate batch effects).

[0098] Analyze the sequencing results: count the number and proportion of reads at each KIR locus (especially KIR2DL4) in each sample; calculate the proportion of KIR2DL4 reads to all functional KIR gene reads; evaluate the reliability of KIR2DL4 genotyping at each proportion (whether it meets the standard of ≥3 reads and ≥99% consistency).

[0099] The results are shown in the table below: Table 4. KIR2DL4 sequencing read percentage and genotyping success rate under different mixing ratios (n=3)

[0100] The results showed that the KIR2DL4 read ratio was too high (42.3%) in the 1:1 ratio, severely crowding out sequencing resources for other KIR genes (especially highly polymorphic genes such as KIR2DL1 and KIR3DL1), resulting in insufficient reads for high-resolution genotyping. While the KIR2DL4 read ratio was slightly higher in the 30:5 and 20:5 ratios than in the 30:2 ratio, the total number of effective reads was lower, and in some samples, the KIR2DL4 read ratio exceeded 20%, indicating insufficient stability. The 30:2 ratio was the most stable among all samples, with an average KIR2DL4 read ratio of 8.2%, significantly reducing its excessive occupation of sequencing resources. At the same time, other KIR genes received sufficient read coverage, resulting in the highest total number of effective reads (mean 92,156), achieving the optimal distribution of sequencing reads.

[0101] Experimental Example 3 Comparative experiments with different primers: To obtain primer combinations capable of amplifying the full-length transcripts of all members of the KIR gene family in a balanced manner, multiple primer pairs were designed in previous studies for all KIR genes except KIR2DL4, as well as for the KIR2DL4 gene itself. Due to the high cDNA sequence homology (over 97%) among members of the KIR gene family, and the unique sequence characteristics of KIR2DL4, designing primers that can specifically distinguish the full-length transcripts of each member is extremely challenging. Conventional primer design software often recommends methods that are unusable due to excessively high off-target rates. Comparative experiments with different primers are shown below.

[0102] (1) Primers for KIR genes other than KIR2DL4 Human peripheral blood total RNA was used as a template (sample numbers 006626 and 006627, with KIR genotypes AB5 and AA1 respectively according to the AFND database standard numbering). Amplification was performed using the FastKing one-step RT-PCR kit (batch number: B0704A) from Tiangen Biotech (Beijing) Co., Ltd., strictly following the manufacturer's instructions. A DL2000 DNA Marker was used (band sizes: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp). The amplification program was as follows: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of annealing at 94℃ for 30 sec, annealing at (65℃, 62℃, 59℃, 56℃) for 30 sec, and annealing at 72℃ for 30 sec; extension at 72℃ for 5 min.

[0103] Amplification tests were performed on 6 pairs of primers at different annealing temperatures. The results are summarized below: Table 5. Comparison of primer results for KIR genes other than KIR2DL4

[0104] Primer pair 6 is the first primer pair of the present invention. The upstream primer sequence (KIR-RNA-TGS-F) is 5'-CCGGCASCACCATGTCRCTC-3', as shown in SEQ ID NO.1, and the downstream primer sequence (KIR-RNA-TGS-R) is 5'-AGAGGTGGGCAGGGGTCAAGT-3', as shown in SEQ ID NO.2.

[0105] Electrophoresis images of the amplification results of 6 primer pairs targeting KIR genes other than KIR2DL4 are shown below. Figure 5 As shown; lane 1 is the marker (DL2000 DNA molecular weight standard, bands from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); lanes 2-5 are the amplification products of sample 006626 (KIR genotype AB5) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃); lanes 6-9 are the amplification products of sample 006627 (KIR genotype AA1) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃).

[0106] The results showed that among the six primer pairs, only primer pair 6 could stably amplify the target band of the expected size (approximately 1.2–1.6 kb) at an annealing temperature of 65 °C without nonspecific amplification. However, at annealing temperatures of 62 °C and lower, the amplification efficiency decreased significantly or nonspecific bands appeared. Among the remaining five primer pairs, one pair (primer pair 3) could amplify the target band, but with severe nonspecificity, while the other four pairs produced no amplification products at all, resulting in a failure rate of 83.3% (5 / 6).

[0107] (2) Primers targeting the KIR2DL4 gene Using human peripheral blood total RNA as templates (sample numbers 006626 and 006627), and employing the same RT-PCR system, primers were designed and tested targeting the KIR2DL4 gene. Amplification was performed at annealing temperatures of 65℃, 62℃, 59℃, and 56℃ for comparison. Two pairs of primers were designed and tested, and the results are summarized below: Table 6. Primer screening results for the KIR2DL4 gene.

[0108] Primer pair 2 is the second primer pair of the present invention. The upstream primer sequence (KIR2DL4-RNA-TGS-F) is 5'-AATGTGTCAACTGCACGATCCGG-3', as shown in SEQ ID NO.3, and the downstream primer sequence (KIR2DL4-RNA-TGS-R) is 5'-GGAAGAGTGATGCTCTAAGATGG-3', as shown in SEQ ID NO.4.

[0109] The electrophoresis results of the two primer pairs targeting KIR2DL4 are shown below. Figure 6 The above describes the following: Lane 1 contains the Marker (DL2000 DNA molecular weight standard, bands from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); Lanes 2-5 contain the amplification products of sample 006626 (KIR genotype AB5) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃); Lanes 6-9 contain the amplification products of sample 006627 (KIR genotype AA1) under different amplification programs (annealing temperatures ① 65℃, ② 62℃, ③ 59℃, ④ 56℃).

[0110] The results showed that, among the two primer pairs, the second primer pair could stably amplify a clear target band of approximately 1.3 kb at an annealing temperature of 59 °C; while the amplification efficiency decreased significantly or the band became blurred at other annealing temperatures, indicating that 59 °C was the optimal annealing temperature for this primer pair. The second primer pair selected in this invention (corresponding to SEQ ID NO. 3~4) can stably and specifically amplify the full-length coding region of KIR2DL4 at an annealing temperature of 59 °C, with a failure rate of 50% (1 / 2).

[0111] The above results demonstrate that the primer combinations described in this invention cannot be easily obtained using conventional PCR primer design strategies. Conventional PCR primer design software (such as Primer-BLAST and Oligo) typically provides multiple candidate schemes at the initial design stage. However, due to the high homology (over 97% sequence similarity between genes) of the KIR gene family addressed in this invention, the conventional primers recommended by the software exhibited extremely high off-target and amplification failure rates in actual amplification. The initial primer design failure rate of up to 75% fully demonstrates that the primer pairs shown in SEQ ID NO.1~SEQ ID NO.4 provided by this invention are different from other conventionally designed primer pairs and can achieve the technical effect of balanced amplification of all members of the KIR gene family.

[0112] Test Example 4 Comparative verification of the method of this invention with existing technical methods.

[0113] To verify the accuracy and superiority of the method of the present invention, peripheral blood samples from 12 healthy individuals were selected, and KIR genotyping was performed using the following three methods, with the results compared: 1. PCR-SSP Method: The KIR Ready Gene SSP kit (batch number: G601020) from Inno-Train (Germany) was used. The kit was operated strictly according to the instructions. The presence / deletion of KIR genes was detected in 12 samples. After PCR amplification products were subjected to 2% agarose gel electrophoresis, the presence (recorded as "1") or deletion (recorded as "0") of each KIR gene was determined according to the interpretation table provided in the instructions.

[0114] 2. DNA-TGS Method (Control Group): Genomic DNA was extracted from the samples. Following the method previously established by our research group [Deng Zhihui, Zhen Jianxin, Ning Li, Li Yunan. Method for Genotyping of Full-Length KIR Genes by Third-Generation Long-Read Sequencing [P]. Guangdong Province: CN120118992A, 2025-06-10.], the samples underwent full-length third-generation sequencing genotyping of KIR genes based on genomic DNA. After constructing the library according to the method published in that literature, CCS mode sequencing was performed using the PacBio Sequel IIe system to obtain HiFi reads (accuracy ≥ Q20). Alignment and allele typing were performed using the IPD-IMGT / KIR database as a reference to obtain allele typing results based on genomic DNA.

[0115] 3. Method of the present invention (RNA-TGS): Following the steps of Experiment Example 1, RNA was extracted from the sample and RT-PCR amplification was performed using the first primer pair and the second primer pair, respectively. The amplification products from the two tubes were mixed at a mass ratio of 30:2. After constructing the library, the PacBio third-generation sequencing platform was used for sequencing to obtain allelic typing results based on the transcriptome.

[0116] The genotyping results of the 12 samples are analyzed as follows: Table 7 Comparison results of three KIR genotyping methods (12 samples)

[0117] Note: ① KIR3DP1 was not detected in RNA-TGS because the downstream primer of the first primer pair could not bind to KIR3DP1, not because the method lacked sensitivity. ② KIR3DL3 and KIR2DP1 were not detected in RNA-TGS because their expression levels in peripheral blood were low (mean TPMs of 3.42 and 68.79, respectively) or they were pseudogenes, which is an inherent characteristic of transcriptome-based methods. ③ Alleles marked with "Mis" indicate unreported missense variations compared to the database reference sequences.

[0118] The results are analyzed as follows: (1) The three methods are completely consistent in terms of the presence / deletion level of major functional KIR genes.

[0119] Allelic typing results from RNA-TGS and DNA-TGS were summarized at the genotypic level (presence / deletion) and compared with those from the PCR-SSP method. The results showed that: For the major functional KIR genes that are truly expressed (including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, and the expressed KIR3DL1), the PCR-SSP results were "1" (present) in all 12 samples. RNA-TGS and DNA-TGS also successfully detected the corresponding alleles. The three methods were completely consistent in determining the presence / deletion of the above genes, demonstrating that the method of this invention has the same reliability as the traditional PCR-SSP method in genotyping at the genotype level.

[0120] For the three genes KIR3DL3, KIR2DP1, and KIR3DP1, PCR-SSP and DNA-TGS detected positive results ("1" or allele), while RNA-TGS did not detect any ("-"). The reasons for this difference are as follows: KIR3DP1 was not detected by RNA-TGS because the downstream primer of the first primer pair could not bind to KIR3DP1 (see Table 1), which is the expected result; KIR2DP1 and KIR3DL3 were not detected, which is an inherent characteristic of transcriptomics methods: KIR2DP1 is a pseudogene and is usually not transcribed and expressed; KIR3DL3 has extremely low expression levels in peripheral blood (average TPM of only 3.42), below the detection threshold.

[0121] (2) RNA-TGS and DNA-TGS are highly consistent at the allele level.

[0122] The allelic typing results of RNA-TGS and DNA-TGS in 12 samples were compared loci one by one. The results showed that the allelic typing results of the two methods were completely identical at all truly expressed functional KIR loci.

[0123] KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DS1: In all 12 samples, the allele types and heterozygous status detected by RNA-TGS and DNA-TGS were completely consistent. This high degree of consistency fully demonstrates the accuracy of the method of this invention in allele-level genotyping, and it has equivalent resolution to third-generation sequencing methods based on gDNA.

[0124] (3) The method of the present invention successfully solves the amplification bias of KIR2DL4.

[0125] In all 12 samples (except for sample 8 which was affected by RNA quality issues), the method of this invention successfully detected the KIR2DL4 allele, and the proportion of KIR2DL4 reads in each sample remained within the ideal range of 5%-15% (average 8.2% ± 1.3%), which is completely consistent with the detection results of the DNA-TGS method. This indicates that the present invention, through the strategy of "pooled amplification + 30:2 optimized mixing", successfully overcomes the technical bottleneck of low KIR2DL4 amplification efficiency and achieves balanced detection of the KIR gene family.

[0126] (4) The inherent characteristics of transcriptomics methods: distinguishing between “genome presence” and “real expression”.

[0127] The method of this invention detects actual KIR transcripts (mRNA) present in the sample, rather than genomic DNA sequences. This technical approach allows it to accurately reflect the actual expression status of genes and automatically filter out genes that are not expressed or are expressed at very low levels, which is one of the core advantages of this invention. The specific analysis of the cases in the table where RNA-TGS was not detected but DNA-TGS / PCR-SSP was detected is as follows: KIR2DP1 and KIR3DP1 (pseudogenes): These genes were not detected by RNA-TGS in all 12 samples. These two genes are internationally recognized pseudogenes. Although they exist intact at the gDNA level, their promoter activity is extremely weak and their mRNA stability is low, typically resulting in no transcriptional expression or extremely low expression levels. The fact that they were not detected in this invention demonstrates that this method successfully avoids the interference of pseudogenes on functional analysis, exhibiting high detection specificity and avoiding the common pitfall of misidentifying pseudogene signals as functional genes.

[0128] KIR3DL3 (low-expression gene): RNA-TGS was not detected in any samples. Existing techniques (based on the GTEx database) indicate that KIR3DL3 has an average TPM of only 3.42 in peripheral blood, indicating extremely low expression levels, below the detection threshold of this invention, which is in line with expectations. This invention only reports the truly expressed transcript, avoiding false positives due to gDNA contamination.

[0129] In summary, the "not detected" results of RNA-TGS for the aforementioned genes are entirely consistent with biological expectations and are an inherent characteristic of transcriptomics methods, rather than a result of insufficient method sensitivity. These results precisely demonstrate that this invention can accurately reflect the expression status of KIR genes in samples, avoid pseudogene interference, and reveal key functional information such as allele expression imbalance.

[0130] This invention maintains the high-resolution advantage of third-generation sequencing while employing a "pooled amplification + optimized mixing" strategy to achieve balanced detection of the KIR gene family (especially KIR2DL4). Compared to the PCR-SSP method, it achieves complete consistency in genotyping of major functional KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and KIR3DS1); compared to the DNA-TGS method, it achieves high consistency in allele levels of truly expressed functional KIR genes. Based on transcriptome detection, this invention accurately reflects the KIR allele composition expressed in samples, automatically eliminates pseudogene interference, and reveals allele expression imbalances, providing reliable technical support superior to traditional gDNA genotyping for KIR gene function research and clinical translation.

[0131] Experimental Example 5 The method of this invention is used to discover potential new allelic variations.

[0132] When performing transcriptome-based KIR genotyping according to the method in Example 4, further analysis of the RNA-TGS sequencing data of samples 5 and 7 revealed two potential novel variants not included in the IPD-IMGT / KIR database (Release 2.15). These variants were confirmed through base-by-base alignment of the sequencing reads with the database reference sequences and Sanger sequencing. The specific variant information is as follows: (1) Sample 5 - KIR2DS4 010 variant (named 2DS4) 010Mis).

[0133] This allele is related to KIR2DS4 in the database. The 00101 reference sequence (GenBank accession number: AY366345) is highly homologous, but contains a missense variant (c.583T>A) at nucleotide 583 (located in the Exon 5 coding region). This variant causes the corresponding codon to change from AGC (encoding serine, Ser) to ACC (encoding threonine, Thr). Both serine and threonine are polar amino acids, but threonine has an additional methyl group on its side chain, which may affect the protein's spatial conformation or interactions with other molecules. A search revealed that this variant site was not found in any known alleles in the IPD-IMGT / KIR database, suggesting it may be a novel allele or a rare population-specific variant.

[0134] (2) Sample 7 - KIR3DL1 00101 variant (named 3DL1) 00101Mis).

[0135] This allele is related to KIR3DL1 in the database. Compared to the reference sequence 00101 (GenBank accession number: AY366362), a missense variant (c.287G>A) exists at nucleotide 287 (located in the Exon 3 coding region). This variant changes the corresponding codon from CGG (encoding arginine, Arg) to CAG (encoding glutamine, Gln). Arginine is a basic, positively charged amino acid, while glutamine is a polar, uncharged amino acid. This change in charge may significantly affect the binding affinity of the KIR3DL1 protein to HLA ligands, thereby influencing NK cell inhibitory signal transduction. A search revealed that this variant site is not found in any known alleles in the IPD-IMGT / KIR database, suggesting it may be a novel allele.

[0136] The discovery of the two aforementioned variant sites demonstrates that the method of this invention can not only be used for routine high-resolution genotyping of KIR genes, but also for the discovery and identification of novel allelic variants. Compared with traditional genotyping methods based on genomic DNA, the transcriptome sequencing-based strategy of this invention can directly capture the truly expressed transcript sequences, avoiding missed detections due to primer mismatches or allelic dropout, and is particularly advantageous for discovering low-frequency or population-specific novel variants. This characteristic provides a powerful tool for KIR genetic diversity research, population evolution analysis, and disease association studies. In summary, the method of this invention exhibits significant advantages in detection accuracy, uniformity, and the ability to discover novel variants, and has promising prospects for scientific research and clinical translation.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer combination for high-resolution typing of the KIR gene based on transcriptome, characterized in that, The primer pair includes a first primer pair and a second primer pair; the upstream primer sequence of the first primer pair is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2; the upstream primer sequence of the second primer pair is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.

4.

2. The primer combination according to claim 1, characterized in that, The first primer pair is used to amplify other KIR genes besides the KIR2DL4 gene, including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1, and KIR2DP1; the second primer pair is used to amplify the KIR2DL4 gene.

3. The application of the primer combination according to any one of claims 1 to 2 in high-resolution typing of the KIR gene.

4. A transcriptome-based KIR genotyping method, said method for non-disease diagnosis or treatment purposes, characterized in that, The method includes the following steps: using sample RNA as a template, reverse transcription PCR amplification is performed using the first primer pair and the second primer pair in any one of the primer combinations described in claims 1 to 2; the first amplification product obtained by amplification using the first primer pair and the second amplification product obtained by amplification using the second primer pair are mixed at a mass ratio of 30:2 to obtain a mixed sequencing library; the mixed sequencing library is sequenced to obtain the sequence information of the KIR gene.

5. The KIR genotyping method according to claim 4, characterized in that, The reverse transcription PCR amplification system includes: 12.5 μl of 2×FastKing One Step RT-PCR Master Mix, 1 μl of 25×RT-PCR Enzyme Mix, 0.8 μl of 10 μM upstream primer, 0.8 μl of 10 μM downstream primer, 5 μl of 40~100 ng / μL RNA template, and 4.9 μl of RNase-Free ddH2O.

6. The KIR genotyping method according to claim 4, characterized in that, The amplification conditions for the first primer pair included: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 65℃ for 30 sec, and 72℃ for 30 sec; and extension at 72℃ for 5 min.

7. The KIR genotyping method according to claim 4, characterized in that, The amplification conditions for the second primer pair included: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 3 min; 35 cycles of 94℃ for 30 sec, 59℃ for 30 sec, and 72℃ for 30 sec; and extension at 72℃ for 5 min.

8. A kit for high-resolution genotyping of the KIR gene, characterized in that, The kit comprises the primer combination as described in any one of claims 1 to 2.

9. The reagent kit according to claim 8, characterized in that, The kit also includes reagents for reverse transcription PCR reactions.

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