P217 Clinical implementation of next-generation DNA sequencing improves high-resolution HLA genotyping

Abstract

Aim Successful hematopoietic stem cell transplantation (HSCT) requires accurate allele-level matching of donor and recipient HLA genes. To improve accuracy, we implemented next-generation DNA sequencing (NGS) methods for high-resolution HLA genotyping of HSCT patients and donors. Here we evaluate NGS HLA typing accuracy and the effect on clinical management of HSCT patients. Methods NGS genotyping for HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 was performed on peripheral blood ( n = 151) and buccal swab ( n = 7) samples for HSCT patients and potential donors between November 2016 and March 2017 using TruSight HLA v2, Nextera XT Index, and MiSeq Nano v2 300 cycle reagents (Illumina). Phased exon sequence data were analyzed using Assign TruSight v2 software. Results HLA typing by NGS resulted in unambiguous allele-level genotyping of 2739/2826 (96.9%) alleles tested. Ambiguities were primarily (71.3%) in DPB1 with inability to discriminate between G group alleles differing in exon 1 or the 5’ UTR. One DPB1 allele had non-G group ambiguity. In 19 alleles ( DRB1, DRB3/4/5, DQA1, DPA1, DPB1 ) no 100% sequence match could be identified in the IMGT reference database. NGS enabled confident identification of 24 non-CWD alleles ( B, DRB1, DPB1 ). The potential clinical impact of this improved accuracy was evaluated by re-analysis of NGS sequencing results using only non-phased DNA sequence for class I exons 2, 3, and 4, and class II exons 2 and 3 to simulate expected results from Sanger-based DNA typing. De-resolution of NGS data for 62 unrelated donors demonstrated ambiguity in 57% of alleles tested. NGS efficiently integrated into the clinical workflow with an average TAT of 5 days and 90.5% of samples meeting the 7 day TAT goal. Buccal swab samples presented difficulty, as 5/7 had to be repeated using increased DNA concentrations to account for DNA degradation. Repeat sequencing successfully genotyped 4/5 buccal swab samples. Conclusions NGS methodology provides significantly improved ability to accurately and unambiguously type HLA genes. Typing accuracy is primarily limited by incomplete reference gene DNA sequence and difficulty in generating full-gene sequence including exon 1 and the 5’ UTR for class II genes.