High-throughput and cost-effective NGS strategy for intermediate-resolution HLA typing

Abstract

Aim The National Marrow Donor Program (NMDP) requires that each newly recruited donor is typed at intermediate resolution level for HLA-A, -B, -C, -DRB1 and optionally -DQB1. Therefore we developed an NGS assay specifically designed for intermediate resolution HLA typing (class I exon 2 & 3, and class II exon 2) that is suitable for high-throughput. PCR conditions were optimized such to reduce the costs of reagents. Moreover, the limited size of the amplicon allows for an increased sample capacity per run on either the MiSeq or Ion Torrent platform: a four-fold increase on average compared to the whole-gene amplification strategy. Method We integrated this NGSessenz strategy into the previously developed NGSgo workflows for MiSeq and Ion Torrent platforms. For the MiSeq platform, high-throughput and reproducibility was verified by running 4 × 96 DNA samples on a micro flow cell. For IonTorrent, we verified the capacity of the smallest (314 chip) and the largest (318 chip) by running a variety of samples. For each sample, loci were pooled prior to the execution of the NGSgo platform-specific library preparation and subsequent NGS procedures were performed. Results We will present the NGS data that were obtained for the robustness of amplification, mappability of the reads, read depth per locus, concordance of NGS typing with reference type, reproducibility of the NGS data generated, and the capacity of the NGSessenz assay on the different platforms. Conclusions Taken together, we will demonstrate the major advantage of this new NGSessenz strategy in ease-of-use and cost-effectiveness for HLA registry typing by means of NGS. I. Van Rooy: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. R. Van Aard: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. B. Luiken: Employee; Company/Organization; GenDx. L. Van de Pasch: Employee; Company/Organization; GenDx. E. Bouwmans: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. W. Mulder: Stock Shareholder; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.