P094 : A FULL NGS WORKFLOW FOR REACHING THE ULTIMATE HLA TYPING RESOLUTION

Aim Currently applied technologies for HLA sequencing-based typing (SBT) often show ambiguous results, most of which are caused by cis–trans allele combinations. In contrast, next-generation sequencing (NGS) allows for reads that originate from a single molecule, meaning that the paternal and maternal alleles can uniquely be identified. As NGS can phase single molecules throughout the gene, HLA typing by means of NGS will yield typing results without ambiguities. The implementation of NGS technology for routine diagnostic purposes would therefore be highly advantageous to obtain reliable, unambiguous HLA typing results. Methods We developed a complete NGS workflow for high-throughput HLA typing compatible with the Illumina NGS platforms. The NGS workflow, including full-length HLA locus amplification, library preparation, and sequencing analysis, is fully optimized to facilitate high-resolution identification of HLA alleles. We generated HLA locus specific amplicons for the class I loci (HLA-A, -B and -C) and class II loci (HLA-DRB1, -DRB3, -DRB4, -DRB5, -DPA1, -DPB1, -DQA1, and -DQB1) on 96 genomic DNA reference samples, using our optimized NGS workflow compatible with the Illumina MiSeq platform. Samples and loci were pooled and run on the MiSeq. Sequence data was analyzed using the NGS HLA typing software NGSengine®. Results Paired-end NGS data was obtained for 11 class I and class II HLA loci pooled for 96 gDNA reference samples in one MiSeq sequencing run. The NGS data covered the complete HLA region of interest by highly accurate measures for all HLA loci tested. All HLA alleles were effectively separated and, in most cases, could be phased throughout the gene, resulting in unambiguous high-resolution HLA typings. High-quality data was obtained for all loci in agreement with the reference SBT allele assignments as provided by Sanger SBT. Conclusion The NGS workflow we have developed demonstrates that it is feasible to perform HLA typing by means of NGS in a reliable, accurate and high-throughput manner that in future will be suitable for routine diagnostic purposes. J. Adema: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. F. Ruzius: Employee; Company/Organization; GenDx. E.H. Rozemuller: Employee; Company/Organization; GenDx. Stock Shareholder; Company/Organization; GenDx. M.T. Penning: Employee; Company/Organization; GenDx. L. van de Pasch: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.