Development of bovine leukocyte antigen – DRB3 genotyping using nanopore sequencing

The bovine leukocyte antigen DRB3 (BoLA-DRB3) gene, a crucial component of major histocompatibility complex class (MHC) II, influences disease susceptibility and production traits in cattle. Conventional PCR – (Sanger)-sequence-based typing (PCR-SBT) – has limitations, including time-intensive processing and phase ambiguity issues. We developed a nanopore-sequencing-based method for BoLA-DRB3 genotyping. The full-length gene (10.9 kbp) was amplified from blood samples of 109 Japanese Black cattle and sequenced using Oxford Nanopore Technologies’ nanopore MinION. The method achieved high-quality metrics, with a mean coverage of 99.97%, an average sequencing depth of 2599× and a mean Q-score of 20.5. Perfect reference sequence alignment was obtained in 92.7% (101/109) of samples. Alleles of all samples showed complete concordance with the set of PCR-SBT typing results for exon 2. The entire process was completed within 3 days (6 h of hands-on time), substantially reducing the time and cost requirements compared with other methods. This nanopore sequencing method provides accurate BoLA-DRB3 genotyping while resolving phase ambiguity through full-length molecule analysis, presenting a potential new standard for bovine MHC genotyping.