Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities
- 270 Downloads
Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.
KeywordsMacaca fascicularis MHC class I PacBio long-amplicon sequencing RNA transcript-based haplotypes
The authors thank Catherine Westbrook, Suzanne Mate, and Galina Koroleva at the US Army Medical Research Institute of Infectious Diseases, and Katherine Munson and the staff of the University of Washington PacBio Sequencing Services core for performing PacBio RS II sequencing. We also thank Annemiek J.M. de Vos-Rouweler for RNA isolations and preparation of cDNAs used in this study. Finally, we thank Nel Otting, Natasja de Groot and the IMGT Non-human Primate Nomenclature Committee for obtaining accession numbers and providing official Mafa transcript nomenclature designations for the sequences described here. This research was supported by contracts HHSN272201600007C and HHSN272201100013C from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, and was conducted at a facility constructed with support from the Research Facilities Improvement Program (RR15459-01, RR020141-01).
- de Groot NG, Otting N, Robinson J, Blancher A, Lafont BA, Marsh SG, O’Connor DH, Shiina T, Walter L, Watkins DI, Bontrop RE (2012) Nomenclature report on the major histocompatibility complex genes and alleles of great ape, old and new world monkey species. Immunogenetics 64(8):615–631CrossRefPubMedPubMedCentralGoogle Scholar
- Shiina T, Yamada Y, Aarnink A, Suzuki S, Masuya A, Ito S, Ido D, Yamanaka H, Iwatani C, Tsuchiya H, Ishigaki H, Itoh Y, Ogasawara K, Kulski JK, Blancher A (2015) Discovery of novel MHC-class I alleles and haplotypes in Filipino cynomolgus macaques (Macaca fascicularis) by pyrosequencing and sanger sequencing : mafa-class I polymorphism. Immunogenetics 67(10):563–578CrossRefPubMedGoogle Scholar
- Wiseman RW, Karl JA, Bimber BN, O’Leary CE, Lank SM, Tuscher JJ, Detmer AM, Bouffard P, Levenkova N, Turcotte CL, Szekeres EJ, Wright C, Harkins T, O’Connor DH (2009) Major histocompatibility complex genotyping with massively parallel pyrosequencing. Nat Med 15(11):1322–1326CrossRefPubMedPubMedCentralGoogle Scholar