中国输血杂志2025，Vol.38Issue(3)：309-315,7.DOI:10.13303/j.cjbt.issn.1004-549x.2025.03.002
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
摘要
Abstract
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Key words
HLA/ambiguities/next generation sequencing/nanopore sequencing technology/third generation sequencing分类
医药卫生引用本文复制引用
陈男英,章伟,董丽娜,王芳,何亿镇,陈晨,朱发明..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[J].中国输血杂志,2025,38(3):309-315,7.基金项目
浙江省自然科学基金(LTGY24H080007)、浙江省医药卫生科学研究基金(2023KY658) （LTGY24H080007）
