NGS HLA typing – Single center experience with Omixon Holotype HLA X4 KIT and HLA twin data analysis

Abstract

Listen

Translate article

Aim Given the high throughput, better coverage, and free of phase ambiguity with HLA typing using next generation sequencing (NGS) technology, we decided to test one of the commercially available kits (Holotype HLA X4 ) offered by Omixon. Methods Total of 32 samples (27 PBMC derived patient DNA samples and 5 UCLA survey DNA samples) were sequenced by NGS for HLA-A, B, C, DRB1 and DQB1 using Holotype HLA X4. All samples were previously typed at high-resolution by combinations of SBT/SSP. This includes 23 HLA-A, 28 B, 21 C, 28 DRB1, 14 DQB1 CWD2.0 alleles and also 2 non-CWD and 2 novel alleles. With Omixon HLA X4 kit, both individual PCR amplicon barcode and pooled amplicon barcode strategies were evaluated on Illumina MiSeq analyzer. Results Long-range PCR gave robust amplifications on all samples for HLA-A, B, C (full coverage) and DRB1 (exon 2–4). DQB1 set 2 PCR (exon 2–5) had a failure rate of 3% (1/32) and DQB1 set 1 PCR (exon 1–4) had a failure rate of 21.9% (7/32, 5 of them from UCLA survey samples). The accuracy of NGS typing was 100% concordant with previous typing for HLA-A, B, C in both PCR amplicon individually barcoded and pooled amplicon barcoded format. Accuracy dropped to 97% for DRB1 and DQB1 assignment in both formats due to technical errors (1/32) or failed DQB1 PCR (1/32). Non-CWD alleles as well as novel alleles were correctly identified. No ambiguities were present in any of the final typing assignments. Using default quality control matrix of 15 parameters from the HLA Twin software, 4/158 resolved HLA alleles gave “red” light in the two traffic light system for amplicon individually barcoded samples, compared to 7/158 alleles in pooled amplicon barcoded samples. Regardless, none of the sample with “red” flagged QC parameter compromised the final HLA typing assignment. Conclusions Our experience with Omixon Holotype HLA X4 kit indicates that HLA-NGS typing indeed has advantages over Sanger SBT method. The Omixon NGS library construction is easy to perform without noticeable bias introduction. The beta version HLA Twin analysis software is user friendly & performs well with both amplicon individually barcoded as well as pooled amplicon barcoded samples with extensive data quality monitoring. However, the robustness of DQB1 PCR requires improvement & the dual algorithm of HLA Twin software needs more transparency.