纳米孔测序技术区分HLA基因分型模棱两可组合结果的探索  

Explore of nanopore sequencing technology in ambiguities of HLA genotyping

作　　者：陈男英[1] 章伟[1] 董丽娜[1] 王芳[1] 何亿镇 陈晨 朱发明[1] CHEN Nanying;ZHANG Wei;DONG Lina;WANG Fang;HE Yizhen;CHEN Chen;ZHU Faming(Blood Center of Zhejiang Province,Hangzhou 310052,China)

机构地区：[1]浙江省血液中心,浙江杭州310052

出　　处：《中国输血杂志》2025年第3期309-315,共7页Chinese Journal of Blood Transfusion

基　　金：浙江省自然科学基金(LTGY24H080007);浙江省医药卫生科学研究基金(2023KY658)。

摘　　要：目的 利用纳米孔测序技术解决新一代测序技术(next generation sequencing, NGS)HLA基因分型中模棱两可组合问题。方法 收集本实验室NGS检测分型存在模棱两可组合的标本38例,采用相同的商品化NGS HLA分型试剂盒引物对HLA-A、-B、-C、-DRB1、-DRB3/4/5、-DQA1、-DQB1、-DPA1和-DPB1位点进行特异性扩增后,实施三代测序建库并在纳米孔测序仪上测序,数据转换成Fastq文件并通过软件指定11个HLA位点基因分型结果。直接计数法统计模棱两可组合结果。结果 以第2域数字作为高分辨结果,三代测序技术(third generation sequencing, TGS)检测的38个标本11个HLA位点结果与NGS结果符合率为100%。TGS法检测HLA-A、-B、-C、-DRB3、-DRB4、-DQA1、-DPA1位点均为唯一性结果,对HLA-A、-B、-C和-DQA1位点模棱两可组合(均因NGS读长短致使定相困难而形成)的区分率为100%;HLA-DRB1、-DRB5、-DQB1和-DPB1位点的模棱两可组合中,TGS法对1号外显子未扩增导致的模棱两可组合的区分率为0%,对因NGS读长短导致的模棱两可组合的区分率为100%。结论 本项目利用纳米孔技术对11个HLA位点模棱两可组合进行区分研究,有效解决了NGS方法短读长引起的模棱两可组合问题,提高了HLA基因分型准确性。Objective To resolve the ambiguities of HLA genotyping generated by next generation sequencing(NGS)using nanopore sequencing technology.Methods A total of 38 samples with ambiguous HLA genotyping by NGS in our la-boratory were collected,and HLA-A,-B,-C,-DRB1,-DRB3/4/5,-DQA1,-DQB1,-DPA1 and-DPB1 loci in these sam-ples were amplified using primers in the same commercial NGS HLA genotyping kit,then subjected to third-generation li-brary construction,and sequenced on the nanopore sequencer.The sequencing data were converted into Fastq files and ana-lyzed by software,and the genotypes of 11 HLA loci were obtained.The ambiguities were counted directly.Results The high-resolution genotyping at the second domain of 11 HLA loci of 38 samples using the third generation sequencing(TGS)were consistent with the results of the NGS method at a rate of 100%.The genotypes for the HLA-A,-B,-C,-DRB3,-DRB4,-DQA1 and-DPA1 loci by TGS were all only one result,and the discrimination rate for ambiguities of the HLA-A,-B,-C,and-DQA1 loci(all caused by the difficulty in phasing due to the short NGS read length)was 100%.Among the HLA-DRB1,-DRB5,-DQB1 and-DPB1 loci,the discrimination rate of TGS for the ambiguities caused by non-amplification of exon 1 was 0%and by the short NGS read length was 100%.Conclusion Nanopore technology was used to identify the ambiguities of 11 HLA loci in this study,and the ambiguities caused by the short read length disadvantage of the NGS meth-od could be solved effectively and the accuracy of HLA genotyping would be improved.

关 键 词：HLA基因 模棱两可 二代测序 纳米孔测序技术 三代测序 

分 类 号：R446[医药卫生—诊断学]