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

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• 作者：Catherine J. Westbrook^a ; Julie A. Karl^b ; Roger W. Wiseman^b ; Suzanne Mate^a ; Galina Koroleva^a ; Karla Garcia^a ; Mariano Sanchez-Lockhart^a ; ^{Mariano.sanchez-lockhart.ctr@mail.mil" class="auth_mail" title="E-mail the corresponding author} ; David H. O&rsquo ; Connor^b ; ^c ; Gustavo Palacios^a
• 关键词：SMRT ; single-molecule real-time ; PacBio ; Pacific Biosciences ; CCS ; circular consensus sequencing ; MCM ; Mauritian cynomolgus macaques ; ChCM ; Chinese cynomolgus macaques ; IPD ; Immuno Polymorphism Database
• 刊名：Human Immunology
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：76
• 期：12
• 页码：891-896
• 全文大小：432 K

文摘

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.