基于Ion Torrent S5平台的二代测序技术对HLA-DRB1基因分型模棱两可进行分析

Analyze the ambiguous results of HLA-DRB1 genotyping using NGS method based on the Ion Torrent S5 platform

目的基于Ion Torrent S5平台NGS法进行HLA-DRB1基因分型检测的模棱两可结果的种类、比例进行分析。方法对471例脐带血造血干细胞库标本采用基于Ion Torrent S5平台的二代测序法检测HLA-DRB1位点,并同步对其中的94例标本采用PCR-SBT测定HLA-DRB1位点,余下377例标本采用PCR-SSO流式磁珠法检测。使用分型软件指定NGS法的HLA-DRB1分型结果,以HLA-DRB1*后第三区的数字作为高分辨水平统计,直接计算法分析模棱两可组合比例。结果标本中470例HLA-DRB1基因NGS检测结果与PCR-SBT法或PCR-SSO法相符合;其中1例标本NGS法漏检一个等位基因,符合率为99.8%。NGS法检测结果中,471例标本中有160例标本HLA-DRB1等位基因型出现模棱两可,比例为33.97%(160/471)。最常见的模棱两可结果组合为DRB1*09∶01∶02/09∶21。结论基于Ion Torrent S5平台的NGS技术可降低HLA-DRB1基因分型的模棱两可结果比例,但仍有一定比例的模棱两可组合结果,同时应注意NGS法检测漏检等位基因的风险。

Objective To analyze the kinds and ratio of ambiguous results of the HLA-DRB1 genotyping using next generation sequencing(NGS) method based on Ion Torrent S5 platform. Methods A total of 471 samples from Blood Center of Zhejiang Province were genotyped for HLA-DRB1 loci by the NGS method,and meanwhile 94 samples among them were detected for HLA-DRB1 loci by PCR-SBT method,and the other 377 samples were detected for HLA-DRB1 loci by PCR-SSO flow fluorescent bead method. The genotyping results of HLA-DRB1 loci by the NGS method were assigned using auto-analytical software. The number of the third field of HLA-DRB1 * was used as high resolution level. The ambiguous combination ratio of the results was calculated by the directly count method. Results The results of NGS detection of HLA-DRB1 gene in 470 samples were consistent with PCR-SBT or PCR-SSO. One allele was missed by NGS method,and the coincidence rate was 99. 8%.Among the 471 samples detected by NGS method,there were 160 samples with ambiguous HLA-DRB1 allelic genotypes,with a proportion of 33. 97%(160/471). The most common combination of ambiguous results is DRB1 * 09 ∶ 01 ∶ 02/09 ∶ 21.Conclusion The NGS technology based on Ion Torrent S5 platform can significantly reduce the ambiguity of HLA-DRB1 genotyping result,while there were still a few HLA-DRB1 genotyping ambiguous results. The risk for allele-dropout should be pay attention in the NGS method.