title: No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.
abstract: Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (∼1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species.
authors.affiliation: U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States.
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Westbrook
authors.affiliation: Wisconsin National Primate Research Center, 555 Science Drive, University of Wisconsin-Madison, Madison, WI 53711, United States.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Karl
authors.affiliation: Wisconsin National Primate Research Center, 555 Science Drive, University of Wisconsin-Madison, Madison, WI 53711, United States.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Wiseman
authors.affiliation: U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Mate
authors.affiliation: U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States.
authors.forename: Galina
authors.initials: G
authors.lastname: Koroleva
authors.affiliation: U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States.
authors.forename: Karla
authors.initials: K
authors.lastname: Garcia
authors.affiliation: U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States. Electronic address: Mariano.sanchez-lockhart.ctr@mail.mil.
authors.forename: Mariano
authors.initials: M
authors.lastname: Sanchez-Lockhart
authors.affiliation: Wisconsin National Primate Research Center, 555 Science Drive, University of Wisconsin-Madison, Madison, WI 53711, United States; Department of Pathology and Laboratory Medicine, 1685 Highland Ave., University of Wisconsin-Madison, Madison, WI 53705, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: O'Connor
authors.affiliation: U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Palacios
mesh_terms: D000483:Alleles; D000818:Animals; D005805:Genes, MHC Class I; D014644:Genetic Variation; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006650:Histocompatibility Testing; D008252:Macaca fascicularis; D017422:Sequence Analysis, DNA
keywords: Cynomolgus macaques; MHC class I; PacBio; Single-molecule real-time circular consensus sequencing (SMRT-CCS)