Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one.
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.
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, 53705, USA.
Dawn M
DM
Dudley
Julie A
JA
Karl
Hannah M
HM
Creager
Patrick S
PS
Bohn
Roger W
RW
Wiseman
David H
DH
O'Connor
D000483:Alleles; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005805:Genes, MHC Class I / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D008253:Macaca mulatta; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; C110764:Mamu-A 01 antigen; C089390:Mamu-A 02 antigen