Aim: To facilitate high-throughput, high-resolution HLA typing, we developed a single-well prototype amplification that enables the generation of sequencing data in <24 hours using Illumina or ONT sequencing. The amplification primers and master mix, which can be used in a total volume as low as 5 µl, were designed to achieve a balanced amplification of all exons of the 11 relevant loci (HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DPB1, DQA1, DPA1) and are feasible for downstream automation.
Methods: Single-well reactions with a total volume of 5 µl were used to analyze 58 diverse cell line samples from the Genetic Testing Reference Material Coordination Program (GeT-RM HLA58). Library preparation was performed using a modified version of the NGSgo® Library Full Kit. Sequencing was performed on a MiniSeq (Illumina) platform and the data was analyzed using an experimental version of NGSengine®.
Results: Sequencing results for all exons of the 11 HLA genes of 58 samples were obtained within one day. 3rd-field typing concordance of 100% with available pre-types was achieved. The sequencing data was of high quality, with a locus mappability of 97.3%, delta Signal-to-Noise of 37.8%, maximum noise of 2.9%, lowest read depth of 544 reads, and estimated second allele of 45.4% (average Exon+ values).
Conclusion: Successful and balanced high-resolution HLA typing was obtained using a prototype single-well amplification assay, using a reaction volume as low as 5 µl. Full exon coverage of all 11 relevant HLA loci (HLA-A, B, C, DRB1, DRB3/4/5, DQB1, DPB1, DQA1, DPA1) was obtained with 100% 3rd-field typing concordance. The future assay is intended to be used in automated systems for high-throughput HLA typing.
