Ambiguous results of the HLA-DPB1 genotyping results: Based on AllType NGS 11-loci sequencing reagent / 中国输血杂志

ABSTRACT

【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.