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

Hiroya Konno1), Jiro Miyamae2), Rei Kajitani3), Kazuto Kugou4), Hiroko Kataoka1), Makoto Akai1), Tomomichi Ishizaka1) and Katsuyoshi Chiba1)

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.

Experimental Animals

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