NDLI: Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one

Content Provider	Springer Nature Link
Author	Dudley, Dawn M. Karl, Julie A. Creager, Hannah M. Bohn, Patrick S. Wiseman, Roger W. O’Conr, David H.
Copyright Year	2013
Abstract	Deep sequencing has revolutionized major histocompatibility complex (MHC) class I analysis of nonhuman primates by enabling high-throughput, economical, and comprehensive genotyping. Full-length MHC class I cDNA sequences, which are required to generate reagents such as MHC–peptide tetramers, cannot be directly obtained by short read deep sequencing. We combined data from two next-generation sequencing platforms to discover novel full-length MHC class I mRNA/cDNA transcripts in Chinese rhesus macaques. We first genotyped macaques by Roche/454 pyrosequencing using a 530-bp amplicon spanning the densely polymorphic exons 2 through 4 of the MHC class I loci that encode the peptide-binding region. We then mapped short paired-end 250 bp Illumina sequence reads spanning the full-length transcript to each 530-bp amplicon at high stringency and used paired-end information to reconstruct full-length allele sequences. We characterized 65 full-length sequences from six Chinese rhesus macaques. Overall, approximately 70 % of the alleles distinguished in these six animals contained new sequence information, including 29 novel transcripts. The flexibility of this approach should make full-length MHC class I allele genotyping accessible for any nonhuman primate population of interest. We are currently optimizing this method for full-length characterization of other highly polymorphic, duplicated loci such as the MHC class II DRB and killer immunoglobulin-like receptors. We anticipate that this method will facilitate rapid expansion and near completion of sequence libraries of polymorphic loci, such as MHC class I, within a few years.
Starting Page	15
Ending Page	24
Page Count	10
File Format	PDF
ISSN	00937711
Journal	Immunogenetics
Volume Number	66
Issue Number	1
e-ISSN	14321211
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-11-16
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Major histocompatibility complex class I Chinese rhesus macaques Roche/454 pyrosequencing Illumina MiSeq sequencing MHC class I genotyping Immunology Human Genetics Gene Function Cell Biology Allergology
Content Type	Text
Resource Type	Article
Subject	Genetics Immunology

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