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.
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Acknowledgments
The authors wish to thank Gabriel Starrett for bioinformatics support. We thank the Batelle Biomedical Research Center for providing us with Chinese rhesus macaque samples. This research was supported by the Department of Health and Human Services Public Health Service grant from the National Institutes of Health under award R24 OD011048.
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The authors declare that they have no conflict of interest.
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Dudley, D.M., Karl, J.A., Creager, H.M. et al. Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one. Immunogenetics 66, 15–24 (2014). https://doi.org/10.1007/s00251-013-0744-3
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DOI: https://doi.org/10.1007/s00251-013-0744-3