Enhanced characterization of 109 genomic dna reference material for 6 HLA loci by next-generation sequencing (NGS)

Provide complete, unambiguous characterization of 6 HLA loci (HLA-A, B, C, DPB1, DQB1, and DRB1) for 109 publicly available cell lines from Coriell Cell Repository, in collaboration with the Center for Disease Control and Prevention’s Genetic Testing Reference Material program (GeT-RM). These cell lines have previously been characterized for 5 pharmacogenetic (PGx) genes (Pratt, V et al J Mol Diag 2010 12(6):835–846) and HLA genes using multiple assays and are intended for use as reference materials for clinical genetic testing laboratories. DNA samples were amplified at 6 HLA loci using Omixon Holotype primers delineating full length genes (5’UTR to 3’UTR) with the exception of DRB1 (intron 1 to intron 4). Sequencing libraries were prepared using Holotype X4 kits for NGS on the Illumina MiSeq. Target (Omixon) and NGSengine (GenDX) were used to analyze the NGS data and results were compared to HLA typing generated by Sanger sequencing (SBT) using SSO and SSP to resolve ambiguities. 187 unique alleles were identified. The table below indicates the percent of people in the given population that will carry one of the alleles included in this study (Maiers, M et al Hum Imunol 2010 68(9):779–788). DPB1 frequencies were not available from this source.Download : Download full-size image We observed 98 5% concordance between HLA typing results using SBT and NGS, with discordance caused by sequence differences outside regions characterized by SBT (n = 21), and errors in SBT/SSP/SSO typing (n = 2). Ambiguities persist in 32/1308 allele calls, with the majority due to alternative cis/trans combinations of exons 2 and 3 in DPB1 (n = 30) and 2 caused by polymorphisms in the unsequenced exon 1 of the DRB1 gene. NGS enhanced the characterization of alleles in this study by identifying errors and allowing analysis of regions not examined by other typing methods. These samples will be useful for assay development and validation, quality control and proficiency testing, and should help to improve the accuracy of PGx and HLA testing in clinical laboratories. D. Monos: Other (Identify); Company/Organization; Royalty.