P082 The robust NGSgo workflow for high-resolution HLA typings also fully compatible with the Ion S5 platform

Abstract

Aim Current technologies for HLA sequencing-based typing often show ambiguous results, most of which are caused by cis–trans allele combinations. Next-generation sequencing (NGS) technology however generates sequences from single DNA molecules, meaning that paternal and maternal alleles can be uniquely identified. Therefore, NGS technology has great potential in the application of HLA typing for diagnostic purposes. The NGSgo ® workflow that proved to be compatible on the Ion PGM platform was now validated on the Ion S5 platform. Here we present NGS data generated when processing pooled HLA loci on the Ion S5 platform using the fully automated Ion Chef system. Methods Amplification of 96 clinical samples was performed for 11 HLA loci with NGSgo-AmpX that enables amplification of whole class I genes, and the essential genomic regions for high-resolution typing of the class II genes. The amplicons generated were pooled per sample and processed in a library preparation that consisted of three steps. The fragmentation and end-repair were performed with the NGSgo-LibrX in a single step followed by the ligation of universal and 24 unique barcoded adapters using the NGSgo-IndX kit (GenDx). Finally, after pooling the libraries in one tube, a magnetic bead step removed all fragments ® software (GenDx). Results Here we will present the data output showing the NGS data quality generated. A preliminary dataset already showed a locus mappability of 89%, read depth of 4206, ambiguities only for 3rd or 4th field resolution (except for DRB1 with 2 ambiguities in the 2nd field due to differences outside of the amplicon) and 100% concordance of the NGS typing with the available pre-types. Conclusions The availability of several NGS workflows allows the end-user to choose the most preferred HLA typing NGS platform that fulfills the requirements of their laboratory for their specific applications. Here we will demonstrate that the NGSgo workflow developed for the Ion PGM is also compatible with the Ion S5 platform, making it a powerful method for high-throughput HLA typings by means of NGS. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx.