P097 : ACCURATE HLA TYPING BY NGS USING THE IonTorrent PGM WITH A PLATFORM SPECIFIC DEVELOPED AND TESTED WORKFLOW

Abstract

Next-generation sequencing (NGS) technology has great potential for the future application of HLA typing in routine diagnostic purposes. NGS sequencing has the advantage that it allows the sequencing of single molecule DNA sequences, meaning that the paternal and maternal alleles can be uniquely identified. As NGS can phase single molecules throughout the gene, HLA typing by means of NGS will yield typing results without ambiguities. Currently, various NGS platforms are available on the market; each platform is based on different technology and each technology has their own specific requirements in order to make them compatible for HLA typing. It is our aim to develop multiple NGS workflows that are each compatible to a specific NGS platform, such as the Illumina MiSeq, the IonTorrent PGM, and the PacBio RS Sequencer. Here we present our latest developments on the IonTorrent PGM platform specific workflow, which is designed for the high-resolution typing of HLA loci. The new IonTorrent workflow for HLA typing includes several consecutive steps, from the most crucial step of whole HLA locus amplification to fragmentation, adaptor ligation, suppression PCR, clonal amplification by emulsion PCR, enrichment of positive ion sphere particles and sequencing on the IonTorrent PGM platform. We present the application of the IonTorrent workflow for 24 samples from standardized genomic DNA reference panels. GenDx adapters are developed and applied, enabling indexing of multiple samples. The NGS data is analyzed using the NGS platform-independent NGSengine® software, developed for NGS HLA sequence analysis (GenDx). The availability of different NGS workflows allows the end-user to choose most preferred SBT-NGS HLA typing method that fulfills their laboratory application needs and requirements. This GenDx NGS workflow for the IonTorrent PGM presented here, is a powerful method to perform HLA typing by means of NGS in a reliable, accurate and high-throughput manner. L. Krol: Employee; Company/Organization; GenDx. J. Adema: 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. L. van de Pasch: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.