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Hum Immunol. 2015 Dec;76(12):891-6. doi: 10.1016/j.humimm.2015.03.022. Epub 2015 May 28.

No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

Author information

1
U.S. Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, United States.
2
Wisconsin National Primate Research Center, 555 Science Drive, University of Wisconsin-Madison, Madison, WI 53711, United States.
3
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.
4
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.

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.

KEYWORDS:

Cynomolgus macaques; MHC class I; PacBio; Single-molecule real-time circular consensus sequencing (SMRT-CCS)

PMID:
26028281
DOI:
10.1016/j.humimm.2015.03.022
[Indexed for MEDLINE]

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