157-P: NGSengine: THE ULTIMATE TOOL FOR NGS HLA TYPING

Aim Sequencing Based Typing (SBT) is the gold standard for high resolution typing, as the technique analyses the actual DNA sequence of the relevant HLA regions. However, Sanger SBT strategies often show ambiguous results, caused by sequencing PCR fragments with a mix of two alleles. Additional sequencing reactions are required to resolve the ambiguities. This can be overcome with Next Generation Sequencing (NGS) technology. One of the unique features of NGS is that reads originate from a single molecule, thus automatically determining the two alleles separately. This allows for development of an HLA genotyping system that yields typing results without ambiguities. In addition, NGS technology is developing fast and has come to a point where it can be applied routinely. Methods We developed NGSengine®, a software package to perform HLA typing based on NGS data. NGSengine® is an integrated approach which includes determining HLA locus, applying a sequence reference, aligning reads, phasing and typing. Since HLA is highly polymorphic, each of these steps contains HLA-specific features. In this updated study we show that HLA typing by NGS can be routinely applied using data of different NGS systems. Results Data was generated on Roche 454, Ion Torrent PGM, PacBio, Illumina MiSeq and HiSeq. All data available from these platforms could be analyzed by NGSengine®. In a single analysis the expected HLA typings were obtained. In case PCR fragments were generated with whole gene amplification (NGS-g o ®) no ambiguities were found since alleles were effectively separated, and the complete gene was covered by the reads. Conclusions NGSengine®: · performs reliable unambiguous HLA allele assignment based on NGS data · enables HLA-typing of NGS data in a single procedure · can be applied on data from different NGS platforms · can be applied on data generated with different amplification strategies · determines parts of allele sequences not yet present in the IMGT/HLA database · is easy to use by ‘single button analysis’. Kooter: GenDx: Employee. Ruzius: GenDx: Employee. Penning: GenDx:Employee. Mulder: GenDx: Employee; Stockholder. Rozemuller: GenDx:Employee; Stockholder.