单分子实时测序在HLA基因分型中的应用初探

Application of single molecule real-time sequencing in HLA genotyping

目的 分析单分子实时(SMRT)测序技术判断HLA基因型、单体型的准确性,以探讨该方法应用于临床的可行性。方法 留取19例需行肾移植的患者血液标本,提取DNA后利用SMRT测序技术检测样本基因序列,并利用PCR-SBT方法验证其准确性。结果 SMRT技术可鉴定出19例样本的HLA基因型和基因单体型,且结果与PCR-SBT方法相符。1和14号样本PCR-SBT方法的C位点结果存在模棱两可现象,结果为HLA-C*03:02/04;03:03/132,SMRT技术可直接区分,结果为HLA-C*03:02:02,03:03:01。另外,SMRT方法在1号样本的B位点发现新的HLA等位基因。结论 SMRT测序技术应用于HLA高分辨率分型具有高度准确性,在HLA单体型分析具有精准定位多个长间距突变位置的独特优势。

Objective To explore the accuracy and the feasibility of this method in clinical application,single molecule real-time(SMRT)sequencing technology was employed to determine HLA genotyping,haploid type.Methods Blood samples were collected from 19 patients who needed kidney transplantation,genomic DNA was extracted respectively,and the sequences of all samples were obtained by SMRT sequencing technology,and then detected by PCR-SBT to verify the accuracy of the result.Results The genotypes and haplotypes of 19 samples could be assigned by SMRT technique,which were consistent with those of the PCR-SBT method.The results of HLA-C locus showed ambiguous combination on samples 1 and 14 using SBT method,with HLA-C*03:02,03:03 and-C*03:04,03:132;while the unique result of them is HLAC*03:02:02,03:03:01 using SMRT technology.In addition,a new HLA-B allele of sample 1 was identified using SMRT method.Conclusion SMRT sequencing technology has high accuracy when applied to HLA high-resolution typing,and has unique advantages of accurately locating multiple long-distance mutation in HLA haploid typing.