부산대학교 도서관

Aim High-resolution HLA typing by DNA sequencing has enabled accurate matching of donors and recipients in allogeneic hematopoietic stem cell transplantation (HSCT). Most laboratories perform high-resolution HLA typing by Sanger-based DNA sequencing methodology. Sequence-based typing (SBT) using this methodology has significant limitations preventing unambiguous HLA typing, often requiring additional testing to provide clinically-useful information. Methods This study examined the potential benefit of next-generation DNA sequencing (NGS) for accurate HLA typing. DNA samples extracted from peripheral blood leukocytes from potential HSCT donors and recipients being tested by SBT for high-resolution HLA typing ( n = 95) were tested in parallel using Illumina TruSight HLA sequencing reagents. In brief, amplicons for HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 were generated by long-range PCR, fragmented and indexed using Nextera XT v2 reagents, and sequenced using a MiSeq Reagent kit v2 (250 cycle paired-end). Data were analyzed blinded using Conexio Genomics Assign software. Results NGS generated unambiguous 2-field high-resolution HLA typing results for 94.2% of alleles typed (Table 1). Ambiguities were clustered, with HLA-B∗44:02/19N, -DPB1∗04:01/105:01 with -DPB1∗04:02/126:01, and HLA-DRB1∗03, -DRB1∗12, and -DRB1∗15 representing 69.4% of all ambiguities. NGS results were 99.8% concordant with SBT. Importantly, NGS resolved ambiguous typings from SBT. Improved resolution was derived from the nearly full-gene sequencing and phased sequence analysis enabled by NGS. Conclusions NGS presents a significant technical advantage for accurate high-resolution HLA typing. The nearly full-gene sequencing combined with phased stranded sequencing is useful for resolving common ambiguities observed using current SBT methodology. N. Baird: Employee; Company/Organization; Illumina, Inc. B. Baas: Employee; Company/Organization; Illumina, Inc. A. Crawford: Employee; Company/Organization; Illumina, Inc. M. Won: Employee; Company/Organization; Illumina, Inc. N. Kim: Employee; Company/Organization; Illumina, Inc. D. Goodridge: Employee; Company/Organization; Conexio Genomics. A. Lindell: Employee; Company/Organization; Illumina, Inc. G.P. Morris: Grant/Research Support; Company/Organization; Illumina. 2. Consultant; Company/Organization; Viracor-IBT Laboratories. 4. Scientific/Medical Advisor; Company/Organization; Neuralstem, Inc. [ABSTRACT FROM AUTHOR]