Aim:
Sequencing based HLA typing generally uses PCR to enrich the HLA loci. However, this can lead to an unbalanced enrichment of alleles and can lead to missing alleles when a sample has a mismatch in the region bound by the PCR primers. Here, hybrid capture methods, instead of PCR, were used to enrich 11 HLA loci (HLA-A, -B, -C, DRB1, DRB3, DRB4, DRB5, DQB1, DQA1, DPB1 and DPA1). Libraries were subsequently sequenced using long-read ONT nanopore sequencing or PacBio SMRT sequencing.
Methods:
Hybrid capture probes designed for the enrichment of 11 HLA genes suitable for subsequent long read sequencing were used to enrich for the MHC region. The obtained libraries were sequenced on both a GridIon (ONT) and Sequel IIe (PacBio). For ONT sequencing, basecalling was performed using the super accuracy method from MinKNOW. Data was analyzed using NGSengine and typing results were compared to pre-typing results.
Results:
We obtained concordant high quality data with both ONT and PacBio sequencing. For both sequencing methods, alleles were well balanced, and no alleles were missed. Additionally, the read depths between loci were well distributed, resulting in a good coverage of all loci. Long read sequencing resulted in fully phased sequences for all loci. Sequencing data obtained full genes, including the complete UTRs of all 11 loci, resulting in the identification of additional variants that would be missed by PCR based methods.
Conclusions:
Hybrid capture can be used in combination with long read sequencing methods to type HLA genes. We obtained whole gene, fully phased, high resolution HLA-typings for 11 loci using both ONT and PacBio sequencing data. While the current panel enriches only 11 HLA loci, it could easily be extended to include additional loci within a single multiplex capture reaction.