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

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 6 Chinese rhesus macaques. Overall, approximately 70% of the alleles distinguished in these 6 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.

KIR3DL01 Recognition of Bw4 Ligands in the Rhesus Macaque: Maintenance of Bw4 Specificity since the Divergence of Apes and Old World Monkeys

“…The Roche/454 sequences were trimmed for quality (error probability limit of 0.01) and then reads >300bp were de novo assembled with 100% minimum overlap identity and 0% mismatches per read using Geneious version 6.1 (43, 44). Consensus sequences generated from the assembly were trimmed with an error probability limit of 0.0001, 0 low quality bases, 0 maximum ambiguities and trimmed for primer sequences.…”

Novel MHC class I full-length allele and haplotype characterization in sooty mangabeys

“…Sample preparation from cDNA to Illumina MiSeq sequencing was performed as previously described (Dudley et al 2014). Fragmented pools of full-length MHC amplicons ranged in average length from 471 to 762bp, determined using the Agilent High Sensitivity Bioanalyzer Kit (Agilent Technologies, Santa Clara, CA, USA).…”

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

“…The advent of massively parallel DNA sequencing technologies has revolutionized the ability to identify the complement of expressed MHC class I alleles in an individual macaque [10][11][12][13]. However, the short read lengths produced by most next-generation sequencing platforms remains a barrier to obtaining the full-length, ~1,100-bp MHC class I open reading frame from a single molecule.…”

“…However, the short read lengths produced by most next-generation sequencing platforms remains a barrier to obtaining the full-length, ~1,100-bp MHC class I open reading frame from a single molecule. MHC class I macaque sequencing assays developed on the Roche/454 platform with a 568-bp cDNA amplicon [12,13] and on the Illumina MiSeq with a 195-bp amplicon [4] provide accurate lineage-level genotyping of macaques by sequencing the highly polymorphic peptide-binding region.…”

“…However, since there are often multiple allelic sequence variants within a lineage group [13,14] and there is evidence that specific allelic variants may have an impact on disease susceptibility and progression [15][16][17], it is important to expand the MHC class I full-length allele databases for macaque animal models. Previously described methods to characterize the full-length MHC class I allele sequences in macaques involve cloning and Sanger-based sequencing cDNA-PCR products [9,18] or assembling overlapping Roche/454 amplicons or fragmented Illumina MiSeq reads extended from a Roche/454 scaffold [11,13]. Although successful, these methods are resource intensive and will be obsolete when Roche ends support of the 454 platform in 2016.…”