PacBioシーケンシングに基づくHLA-DRB1遺伝子型判定における曖昧な結果の解析と確認

To analyze and confirm the ambiguous results of HLA-DRB1 genotyping in one case.

HLA genotyping was performed on a sample of hematopoietic stem cell donor using Illumina MiSeq-based next-generation sequencing (NGS). The ambiguous results of HLA-DRB1 locus were further analyzed and confirmed through PacBio SMRT third-generation sequencing.

The Illumina MiSeq-based NGS typing results suggested the presence of a new HLA-DRB1*11 allele (DRB1*11:NEW, 12:01) in the specimen, with a mismatch of G>A located in the 40^th residue of exon 1 compared with the nearest allele DRB1*11:01:01:03. However, due to the long sequence of intron 1, this observed mutation site was so far away from the near heterozygous sites that no reads could cover this gap. Therefore, it was impossible to determine which consensus the mutation site was located in, and the NGS-based genotyping results were obtained from the random allocation by the software, which was ambiguous and unreliable. In order to confirm the results, the long-read third generation sequencing technology based on PacBio was applied to genotype the DRB1 locus. The results showed that the DRB1 typing was HLA-DRB1*11:01,12:10. E1-40A was actually located in the allele HLA-DRB1*12:XX , which was exactly matched with HLA-DRB1*12:10.

For some new alleles suggested by NGS, especially the ambiguous ones that are far away from other heterozygous sites, it is necessary to analyze and confirm them by other methods such as the third-generation long-read sequencing technology to obtain reliable results.