P008 Ashi validation with holotype HLA for high resolution typing

Abstract

Aim Clinical validation of Next Generation sequencing(NGS) for HLA typing has been a high priority for many HLA laboratories worldwide, giving the opportunity to assess the correct allele matching of high-resolution genotypes to improve transplantation outcomes and decrease the rate of GvHD.Holotype HLA (Omixon Inc.) is an optimized, pre-configured assay and software combination product that provides comprehensive gene characterization of multiple HLA loci for sequencing on the Illumina platform. The goal of this study is to provide a validation of high-resolution HLA typing using Omixon’s NGS kits. Methods To test the robustness of the NGS protocol for HLA typing by Omixon Holotype HLA Assay and validate its use in our lab, we will type 96 DNA samples from peripheral blood using an 11-locus kit configuration to genotype HLA class I and Class II genes. All DNA samples have been previously typed by Sanger sequencing. HLA typing will be assigned using Omixon’s HLA Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Inter-laboratory reproducibility will be assessed with 20 samples from Children’s Hospital of Philadelphia, Immunogenetics Laboratory, as a courtesy from Dr. Dimitri Monos. The validation will follow the Minimum Guidelines for Validation of NGS for HLA standards from American Society for Histocompatibility and Immunogenetics (ASHI). Results Sequence concordance will be assessed, where allele calls (1st and 2nd fields) from each sample will be compared with those obtained by Sanger sequencing. Reproducibility between technologists will be also assessed. We expect to demonstrate the advantages of the NGS-based HLA genotyping over SSO and SBT techniques, as it may provide credible and more informative HLA genotyping with the first passage without any reflexive testing. Conclusions It’s expected fully concordance of sequencing results for allele call for first and second field. Concluding this validation will provide the confidence that HLA typing by NGS is ready to be introduced in our laboratory. According to our best knowledge, this will be the first validation using Omixon Holotype Assay in Brazil, and the second laboratory in Brazil to validate NGS following the ASHI standards.