High-resolution HLA typings obtained for high-throughput NGS using the Illumina MiSeq platform

Aim Next-generation sequencing (NGS) technology generates sequences from single DNA molecules that enables unique identification of the paternal and maternal alleles. The NGS technology has great potential in the application of HLA typing for diagnostic purposes and generating reliable, unambiguous HLA typing results in a high-throughput fashion. Here we present data obtained for 11 HLA loci using a HLA typing assay dedicated for high-throughput NGS-based HLA typing on the Illumina MiSeq. The implementation of this high-throughput NGS strategy would be highly advantageous to obtain robust high-resolution HLA typing results for registry labs and routine diagnostic purposes. Method A large genomic DNA reference panel, including UCLA reference samples, were selected for high-throughput NGS HLA typing. The whole gene of HLA-A, -B, -C, and the essential genomic regions of HLA-DRB1, -DRB345, -DQA1, -DQB1, -DPA1 and -DPB1 were PCR-amplified in a single PCR for each locus. Amplicons of each sample were pooled and processed using our NGSgo® DNA library preparation workflow for Illumina MiSeq. During library preparation the samples were fragmented, barcoded and multiplexed. Finally paired-end sequencing (2x150 cycles) was performed on the Illumina MiSeq platform using a standard flow cell and data was analysed using our HLA typing software NGSengine®. Results Paired-end NGS data was obtained for 11 HLA loci (class I and II) of most genomic DNA reference samples in a single Illumina 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. For the majority of samples, unambiguous third- and fourth-field resolution typing were obtained for all loci that are in concordance with the second-field or higher reference types. Conclusions The high-throughput NGSgo® strategy that we have developed demonstrates to be a powerful method to perform high-resolution HLA typing by means of NGS in a robust and reliable manner. L. Van de Pasch: Employee; Company/Organization; GenDx. M. Bacelar: Employee; Company/Organization; GenDx. C. Rebel: Employee; Company/Organization; GenDx. R. Van Aard: Employee; Company/Organization; GenDx. R. Kooter: Employee; Company/Organization; GenDx. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx. E. Bouwmans: Employee; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx.