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

Area of Science:

• Veterinary Genetics
• Immunogenetics
• Next-Generation Sequencing

Background:

• Bovine leukocyte antigen (BoLA) genes are highly polymorphic and exhibit copy number variations (CNV), complicating genome-wide resequencing.
• Accurate genotyping of BoLA is crucial for cattle breeding and disease resistance studies.

Purpose of the Study:

• To develop and validate a hybridization capture target next-generation sequencing (NGS) method for efficient BoLA region resequencing.
• To assess the utility of this method for identifying single nucleotide polymorphisms (SNPs), indels, and CNVs in BoLA genes.

Main Methods:

• Design of custom probe sets for hybridization capture of BoLA DNA sequences.
• Construction of DNA libraries and enrichment using targeted capture sequencing.
• Genotyping of BoLA-DRB3 using both the developed NGS method and traditional PCR sequence-based typing (SBT).
• Analysis of SNPs, indels, and CNVs from sequencing data.

Main Results:

• The developed probe set (BoLA2) achieved high coverage (90.8%) and depth (108 reads) for BoLA resequencing.
• Identified 113,646 SNPs and 17,995 indels, with high concordance for BoLA-DRB3 genotyping compared to PCR-SBT.
• Enabled CNV analysis for multiple BoLA class I and II genes, facilitating haplotype definition.

Conclusions:

• Hybridization capture target NGS is a cost-effective and accurate strategy for high-throughput genotyping of the complex BoLA region.
• This method facilitates the study of genetic diversity, CNVs, and haplotypes in cattle.
• The approach is suitable for large-scale genetic research in bovine populations.