AllType NGS11位点测序试剂在HLA-DPB1基因分型中的模棱两可结果分析

Ambiguous results of the HLA-DPB1 genotyping results: Based on AllType NGS 11-loci sequencing reagent

目的 统计分析AllType NGS 11位点测序试剂在Ion Torrent S5和Illumina Miseq 2种二代测序平台对HLA-DPB1基因进行分型检测时的模棱两可结果情况。方法 采用One Lambda公司AllType NGS 11位点测序试剂盒检测434个患者或供者标本的HLA-DPB1基因,其中在Ion Torrent S5测序平台上检测了336个标本,在Illumina Miseq测序平台上检测了98个标本;并同步对434份标本采用PCR-SSO流式磁珠法复核HLA-DPB1基因。对HLA-DPB1*13∶01∶01/107∶01模棱两可等位基因,采用Sanger测序法检测区分。使用TypeStream Visual专业软件指定NGS法的HLA-DPB1基因分型结果,直接计数法统计分析模棱两可组合比例。结果 NGS法以HLA-DPB1命名*后第3区域数字作为高分辨结果进行统计,434个标本中共有357个标本出现模棱两可结果,占82.3%(357/434);其中Ion Torrent S5测序平台336个标本有275个标本存在模棱两可结果,占81.8%(275/336),表现出45种类型;Illumina Miseq测序平台98个标本有82个标本存在模棱两可结果,占83.7%(82/98),表现出27种类型。所有标本HLA-DPB1基因经PCR-SSO法复核,未发现NGS法漏检HLA-DPB1等位基因情况。Sanger测序法检测区分41个标本,共43个HLA-DPB1*13∶01∶01/107∶01模棱两可的等位基因,发现HLA-DPB1*13∶01∶01等位基因为25个,占58.1%(25/43);HLA-DPB1*107∶01等位基因为18个,占41.9%(18/43)。结论 应用AllType NGS 11位点测序试剂仍有较高比例的HLA-DPB1基因模棱两可组合结果。利用Sanger测序法分析HLA-DPB1基因1号外显子可区分解决部分HLA-DPB1*13∶01∶01/107∶01模棱两可等位基因。

Objective To statistically analyze the characteristics of ambiguous results of HLA-DPB1 genotyping given by AllType NGS 11-loci sequencing reagent via two next generation sequencing platforms, i.e. Ion Torrent S5 and Illumina Miseq. Methods A total of 434 samples from patients or donors were genotyped for HLA-DPB1 locus using AllType NGS 11-loci sequencing reagent from One Lambda company;336 samples of them were sequenced via the Ion Torrent S5 platform and other 98 samples were sequenced via the Illumina Miseq platform. All 434 samples were genotyped for HLA-DPB1 gene simultaneously using PCR-SSO flow fluorescent bead method. The ambiguous genotypes of HLA-DPB1*13∶01∶01/107∶01 were distinguished by Sanger sequencing. The HLA-DPB1 genotype results by NGS method were assigned by TypeStream Visual professional software, and the ratio of ambiguous combination was calculated by direct count method. Results Ambiguous results were found in 357 out of 434 samples, accounting for 82.3%(357/434) when HLA-DPB1 allele was assigned to the third field using NGS method. Ambiguous results with 45 types were given in 275 out of 336 samples by the Ion Torrent S5 platform, accounting for 81.8%(275/336) and 82(with 27 types) out of 98 samples by the Illumina Miseq platform, accounting for 83.7%(82/98). All samples were re-genotyped for HLA-DPB1 gene by PCR-SSO, and none HLA-DPB1 allele had been missed by NGS. A total of 43 ambiguous alleles in HLA-DPB1*13∶01∶01/107∶01 involving 41 samples were distinguished by Sanger sequencing;HLA-DPB1*13∶01∶01 were detected in 25(58.1%, 25/43) and HLA-DPB1*107∶01 in 18(41.9%, 18/43). Conclusion There were still a high proportion of HLA-DPB1 ambiguous combinations using the AllType NGS 11-loci sequencing reagent. Sequencing exon 1 of HLA-DPB1 gene by Sanger sequencing can resolve part of the ambiguous results in HLA-DPB1*13∶01∶01/107∶01 alleles.