Complete sequencing of a cynomolgus macaque major histocompatibility complex haplotype

Abstract

Macaques provide the most widely used nonhuman primate models for studying immunology and pathogenesis of human diseases. While the macaque major histocompatibility complex (MHC) region shares most features with the human leukocyte antigen (HLA) region, macaques have an expanded repertoire of MHC class I genes. Although a chimera of two rhesus macaque MHC haplotypes was first published in 2004, the structural diversity of MHC genomic organization in macaques remains poorly understood due to a lack of adequate genomic reference sequences. We used ultra-long Oxford Nanopore and high-accuracy PacBio HiFi sequences to fully assemble the ∼5.2 Mb M3 haplotype of an MHC-homozygous, Mauritian-origin cynomolgus macaque (Macaca fascicularis). The MHC homozygosity allowed us to assemble a single MHC haplotype unambiguously and avoid chimeric assemblies that hampered previous efforts to characterize this exceptionally complex genomic region in macaques. The high quality of this new assembly is exemplified by the identification of an extended cluster of sixMafa-AGgenes that contains a recent duplication with a remarkably similar ∼48.5 kb block of sequence. The MHC class II region of this M3 haplotype is similar to the previously sequenced rhesus macaque haplotype and HLA class II haplotypes. The MHC class I region, in contrast, contains 13MHC-Bgenes, fourMHC-Agenes, and threeMHC-Egenes (versus 19MHC-B, twoMHC-A, and oneMHC-Ein the previously sequenced haplotype). These results provide an unambiguously assembled single contiguous cynomolgus macaque MHC haplotype with fully curated gene annotations that will inform infectious disease and transplantation research.

Complete genomic assembly of Mauritian cynomolgus macaque killer immunoglobulin-like receptor and natural killer group 2 haplotypes

“…High molecular weight (HMW) DNA was prepared as previously described (22). DNA was extracted from ~5×10 6 PBMCs using New England Biolab's Monarch high-molecular-weight DNA extraction kit, following the manufacturer's protocol.…”

“…However, this requires the development of comprehensive characterizations of the key immune-related gene families to develop high throughput and cost-effective genotyping strategies. As mentioned earlier, we previously developed a hybrid, long-read assembly approach to resolve an MCM MHC region (22). Here, we extend this methodology to assemble KIR and NKG2 genomic regions from thirteen representative MCM individuals.…”

“…HLA haplotypes contain three highly polymorphic class I genes: HLA-A, HLA-B, and HLA-C. In contrast, the MHC region of macaques has undergone intricate duplications, deletions, and rearrangements such that each haplotype has gained variable numbers of less polymorphic MHC-A and MHC-B loci (21)(22)(23). A similar level of genetic complexity extends into MHC-related gene families.…”

“…Recently, long-read techniques have been used to resolve MHC and KIR genomic haplotypes from macaques. We previously described a comprehensive five-megabase pair (Mb) MCM MHC genomic assembly using a hybrid sequencing technique (22). In that effort, we combined sequencing technologies, capitalizing on the structural insights of ONT ultra-long reads, and the precision of PacBio high fidelity (HiFi) reads to define the MHC region accurately.…”