title: Large-scale genotyping of highly polymorphic loci by next-generation sequencing: how to overcome the challenges to reliably genotype individuals?
abstract: Studying the different roles of adaptive genes is still a challenge in evolutionary ecology and requires reliable genotyping of large numbers of individuals. Next-generation sequencing (NGS) techniques enable such large-scale sequencing, but stringent data processing is required. Here, we develop an easy to use methodology to process amplicon-based NGS data and we apply this methodology to reliably genotype four major histocompatibility complex (MHC) loci belonging to MHC class I and II of Alpine marmots (Marmota marmota). Our post-processing methodology allowed us to increase the number of retained reads. The quality of genotype assignment was further assessed using three independent validation procedures. A total of 3069 high-quality MHC genotypes were obtained at four MHC loci for 863 Alpine marmots with a genotype assignment error rate estimated as 0.21%. The proposed methodology could be applied to any genetic system and any organism, except when extensive copy-number variation occurs (that is, genes with a variable number of copies in the genotype of an individual). Our results highlight the potential of amplicon-based NGS techniques combined with adequate post-processing to obtain the large-scale highly reliable genotypes needed to understand the evolution of highly polymorphic functional genes.
authors.affiliation: 1] Laboratoire Biométrie et Biologie Evolutive, Université de Lyon, CNRS, UMR5558, Université Lyon 1, F-69622, Villeurbanne, F-69000 Lyon, France [2] Université Lyon, VetAgro Sup Campus Vet, Marcy-L'Étoile, France.
authors.forename: M
authors.initials: M
authors.lastname: Ferrandiz-Rovira
authors.affiliation: Laboratoire Biométrie et Biologie Evolutive, Université de Lyon, CNRS, UMR5558, Université Lyon 1, F-69622, Villeurbanne, F-69000 Lyon, France.
authors.forename: T
authors.identifier: 0000000185044675
authors.initials: T
authors.lastname: Bigot
authors.affiliation: Laboratoire Biométrie et Biologie Evolutive, Université de Lyon, CNRS, UMR5558, Université Lyon 1, F-69622, Villeurbanne, F-69000 Lyon, France.
authors.forename: D
authors.initials: D
authors.lastname: Allainé
authors.affiliation: 1] Laboratoire Biométrie et Biologie Evolutive, Université de Lyon, CNRS, UMR5558, Université Lyon 1, F-69622, Villeurbanne, F-69000 Lyon, France [2] Université Lyon, VetAgro Sup Campus Vet, Marcy-L'Étoile, France.
authors.forename: M-P
authors.initials: MP
authors.lastname: Callait-Cardinal
authors.affiliation: Laboratoire Biométrie et Biologie Evolutive, Université de Lyon, CNRS, UMR5558, Université Lyon 1, F-69622, Villeurbanne, F-69000 Lyon, France.
authors.forename: A
authors.initials: A
authors.lastname: Cohas
mesh_terms: D000483:Alleles; D000818:Animals; D005805:Genes, MHC Class I; D005802:Genes, MHC Class II; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008392:Marmota / Q000235:genetics; D018895:Microsatellite Repeats; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods