Abstract
The aim of this study was to evaluate the workflow and performance of a NGS-based high resolution HLA typing prototype assay developed by GenDx. 48 UCLA reference panel samples were amplified at 11 loci (HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DQA1, DPB, DPA) using NGS-go® primers with Long-Range (LR) PCR and sequenced on the Illumina MiSeq platform. A full gene (5’ UTR to 3’ UTR) amplification strategy was applied with the exception of DRB1 which amplified from exon 2 to exon 4. Individual amplicons were generated for each sample using 100 ng of gDNA per LR-PCR reaction. Intrapatient amplicons were pooled, enzymatically fragmented, and purified. Purified fragmented dsDNA was brought through library preparation using NEBNext (New England Biolabs) for MiSeq sequencing. Due to the limited number of indices (24) in NEBNext, two 2 × 150 paired-end sequencing runs were completed with each run containing 24 samples. Demultiplexed reads were aligned and typed using GenDx NGSengine® v1.4.0 (IMGT v 3.15.0) and Omixon Target v1.8.0 (IMGT v 3.10.0) software. 47/48 UCLA samples successfully amplified; a single sample failed potentially due to gDNA integrity. For HLA-A, B, C, DRB1, DQB1, DQA1, DPB and DPA an average of 5μg of dsDNA was produced per amplicon, and 99.7% agreed with the reference SBT allele assignments. In one sample DRB1 04:05/15:02 were mistyped by both software packages. SBT reference typing was not available for DRB3/4/5, however when both software packages called a loci, there was 100% agreement at 4 digit allele typing level. Average sequencing output was 3.65 Gb of sequence (12 million paired sequence reads) per run and a mean of 459,573 (median: 433,292) paired-end reads per sample. We have demonstrated that GenDx’s NGS HLA typing assay has high accuracy, throughput, and provided unambiguous high resolution results for 47/48 UCLA reference panel samples. This assay delivered full genomic sequences for all targeted HLA loci except DRB1 exon 1 and had the capacity to analyze 11 loci for 24 samples per MiSeq run.
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