PacBio long-read amplicon sequencing of dog leukocyte antigen genes at full-length level in beagle dogs

The major histocompatibility complex (MHC) plays a critical role in individual immune responses and susceptibility to various conditions, including autoimmune diseases and drug reactions. In dogs, the canine MHC (dog leukocyte antigen, DLA) polymorphism is key to understanding immune mechanisms, but technical challenges have impeded its comprehensive genetic analysis. This study addressed these issues by using a novel DLA genotyping method combining long-range PCR and PacBio single-molecule real-time sequencing to analyze the full-length DLA class I and II gene sequences in 83 beagle dogs from two different strains (TOYO and Marshall), which are commonly used as laboratory animals. As a result of genotyping using the full-length sequences, 9, 5, 2, 6, and 8 extended alleles were newly discovered for the DLA class I genes in DLA-88, DLA-12, DLA-88L, DLA-64, and DLA-79, respectively. For the DLA class II genes, 11, 18, 12, and 8 extended alleles were newly discovered in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. There were 25 haplotypes consisting of extended alleles, in contrast to only 10 haplotypes classified using only peptide binding site sequences. Furthermore, comparisons between the strains revealed differences in haplotype frequencies and genetic differentiation. The full-length analysis also provided preliminary insights into regulatory elements, such as promoter and CpG island polymorphisms in DLA-DQB1. The results of this research have important implications for the understanding of the relationship between DLA polymorphism at full length and individual immune responses in dogs.