Four Target Resequencing for the Bovine Major Histocompatibility Complex Region. Proof of Concept

The bovine leukocyte antigen (BoLA) comprises four regions that contain a high density of polymorphic genes and frequently show gene copy number variations (CNV). Therefore, genotyping BoLA using genome-wide resequencing is difficult. This study aimed to develop four probe sets for resequencing of the BoLA region using a hybridization capture target next-generation sequencing (NGS) method. This proof of concept showed and discussed the several applications of the used strategy. DNAs from nine Japanese Black cows and one Holstein cow were genotyped for BoLA-DRB3 using PCR sequence-based typing (SBT). DNA libraries were constructed using the KAPA HyperPlus Kit, and BoLA DNA sequences were enriched using the SeqCap EZ kit and four custom-made probes. Based on preliminary results, the probe set BoLA2 was selected for further analysis. This analysis resulted in a mean coverage of 90.8% with an average depth of 108 reads. A total of 113,646 SNPs and 17,995 indels were detected, several of which have previously been described in the dbSNP database. This allowed the genotyping of class II genes, including BoLA-DRB3. A comparison between target resequencing and PCR-SBT assays did not show conflicts between the BoLA-DRB3 genotyping results. CNV analysis based on read number inferred that the BoLA-DQA1, BoLA-DQA2, BoLA-DQA5, and BoLA-DQB genes would be present in the homozygous or heterozygous states or absent, allowing for the definition of four class II and three class I haplotypes. In addition, CNV of non-classical class I genes were also observed. In conclusion, results show that approach used in this study is a cost-effective strategy for sequencing large samples for many research purposes.