P160 Development of a multiplexed assay and software package for HLA disease association screening with NGS

Aim HLA genes are the most polymorphic genes in the human genome. The encoded HLA molecules play a pivotal role in generating a target dependent immune response. Several HLA molecules are even associated with a specific disease or drug responsiveness. For example; HLA-B∗27 is associated with Ankylosing Spondilitis, HLA-B∗57:01 is associated with abacavir hypersensitivity, and the HLA-DQ2 and HLA-DQ8 haplotypes are associated with Celiac Disease. Prototypes of the assay and software were tested in an Early Access Program (EAP). Methods We have developed a single tube multiplexed amplification assay for fast and cost-effective genotyping of HLA-related diseases based on exon 2 and exon 3 of HLA-B, HLA-DQA1 and HLA-DQB1 genes (NGSgo-HLA-LinkX). Amplicons were processed in our NGSgo workflow for Illumina, and the resulting library was sequenced on the Illumina MiSeq. Obtained sequencing data was analyzed in our newly developed software package LINKengine, which reports whether a sample is positive or negative for the specific HLA allele. Furthermore, LINKengine reports the reliability of its results. Results Here we present the first results of the Early Access Program, including findings on the amplification protocol as well as details of the analyzed data generated for HLA-B, HLA-DQA and HLA-DQB. Conclusions The reagents in combination with the software provide an easy method to genotype disease-related HLA genes. As the workflow after amplification is identical to the workflow of other NGSgo amplified products, amplicons can be combined in a single sequencing experiment, enabling more efficient use of the capacity of the NGS sequencing equipment.