title: Next-generation genotyping of hypervariable loci in many individuals of a non-model species: technical and theoretical implications.
abstract: BACKGROUND
  Across species, diversity at the Major Histocompatibility Complex (MHC) is critical to disease resistance and population health; however, use of MHC diversity to quantify the genetic health of populations has been hampered by the extreme variation found in MHC genes. Next generation sequencing (NGS) technology generates sufficient data to genotype even the most diverse species, but workflows for distinguishing artifacts from alleles are still under development. We used NGS to evaluate the MHC diversity of over 300 captive and wild ring-tailed lemurs (Lemur catta: Primates: Mammalia). We modified a published workflow to address errors that arise from deep sequencing individuals and tested for evidence of selection at the most diverse MHC genes.
  
  
  RESULTS
  In addition to evaluating the accuracy of 454 Titanium and Ion Torrent PGM for genotyping large populations at hypervariable genes, we suggested modifications to improve current methods of allele calling. Using these modifications, we genotyped 302 out of 319 individuals, obtaining an average sequencing depth of over 1000 reads per amplicon. We identified 55 MHC-DRB alleles, 51 of which were previously undescribed, and provide the first sequences of five additional MHC genes: DOA, DOB, DPA, DQA, and DRA. The additional five MHC genes had one or two alleles each with little sequence variation; however, the 55 MHC-DRB alleles showed a high dN/dS ratio and trans-species polymorphism, indicating a history of positive selection. Because each individual possessed 1-7 MHC-DRB alleles, we suggest that ring-tailed lemurs have four, putatively functional, MHC-DRB copies.
  
  
  CONCLUSIONS
  In the future, accurate genotyping methods for NGS data will be critical to assessing genetic variation in non-model species. We recommend that future NGS studies increase the proportion of replicated samples, both within and across platforms, particularly for hypervariable genes like the MHC. Quantifying MHC diversity within non-model species is the first step to assessing the relationship of genetic diversity at functional loci to individual fitness and population viability. Owing to MHC-DRB diversity and copy number, ring-tailed lemurs may serve as an ideal model for estimating the interaction between genetic diversity, fitness, and environment, especially regarding endangered species.
authors.affiliation: University Program in Ecology, Duke University, Durham, NC, USA. kathleen.e.grogan@gmail.com.|Department of Evolutionary Anthropology, Duke University, Durham, NC, USA. kathleen.e.grogan@gmail.com.|Emory University, Room 2006 O. Wayne Rollins Research Center, 1510 Clifton Rd NE, Atlanta, GA, 30322, USA. kathleen.e.grogan@gmail.com.
authors.forename: Kathleen E
authors.initials: KE
authors.lastname: Grogan
authors.affiliation: Department of Evolutionary Anthropology, Duke University, Durham, NC, USA.
authors.forename: Gwendolyn J
authors.initials: GJ
authors.lastname: McGinnis
authors.affiliation: Department of Anthropology, University of Colorado-Boulder, Boulder, CO, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Sauther
authors.affiliation: Department of Anthropology, University of North Dakota, Grand Forks, ND, USA.
authors.forename: Frank P
authors.initials: FP
authors.lastname: Cuozzo
authors.affiliation: University Program in Ecology, Duke University, Durham, NC, USA.|Department of Evolutionary Anthropology, Duke University, Durham, NC, USA.|Department of Biology, Duke University, Durham, USA.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Drea
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D007900:Lemur / Q000235:genetics*; D008285:Major Histocompatibility Complex / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA