P143 Incorrect allele assignment by Sanger is novel allele by NGS

Aim The human leukocyte antigen (HLA) gene family encodes the major histocompatibility complex (MHC) proteins in humans. Being the most polymorphic region of the human genome, it has proven difficult to fully characterize. NGS has shown that it is able overcome this challenge and provide unambiguous and accurate HLA typings. NGS typing of all patients and donors is now standard practice for our center. Previously, high resolution typing would be provided by Sanger sequencing (SBT) with many ambiguous allele pairs ruled out by RSSOP. This study seeks to show the difference in typing assignments between our previous strategy of combined SBT & RSSOP vs typing by NGS for two proficiency samples. Methods We typed two proficiency samples by SBT/RSSOP and NGS. This provided us with the typing resolution that we previously would have reported. These same samples were tested by NGS using Omixon’s Holotype HLA kit. A DNA library was created by pooling 24 samples total and sequenced on the Illumina Miseq using a Micro Kit. NGS analysis was performed with Omixon’s HLA Twin 2.1.0. Results NGS provided a higher resolution and less ambiguous typing than SBT/RSSOP. In Sample 1, the HLA typing was not A∗02:380, A∗30:01:01, but in fact A∗02:01:01 and a novel variant of A∗30:01:01. The variant produced a missense SNP in codon 139 from GCG to GTG which resulted in an amino acid change from alanine to valine. In Sample 2, with SBT/RSSOP, we were not able to obtain a common 4 digit typing for HLA-B and therefore were not sure if the B∗15 or B∗46 was the common allele. NGS was able to unambiguously assign B∗15:58, B∗46:01:01. Conclusions SBT/RSSOP was not able to provide the correct typing for HLA-A for Sample 1. By utilizing NGS, we were able to determine that this sample had a novel allele that was not characterized by SBT/RSSOP. In addition, NGS has been useful for us in obtaining more accurate and unambiguous typings as can be seen in both samples described here. Download high-res image (236KB) Download full-size image