title: Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird.
abstract: Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.
authors.affiliation: Institute of Nature Conservation, Polish Academy of Sciences, Al. Mickiewicza 33, 31-120, Kraków, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Biedrzycka
authors.affiliation: Evolutionary Biology Group, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, 61-614, Poznań, Poland.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sebastian
authors.affiliation: Evolutionary Biology Group, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, 61-614, Poznań, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Migalska
authors.affiliation: Department of Biology, Lund University, Ecology Building, Sölvegatan 37, 223 62, Lund, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Westerdahl
authors.affiliation: Evolutionary Biology Group, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, 61-614, Poznań, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Radwan
mesh_terms: D000818:Animals; D005805:Genes, MHC Class I; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D046109:Passeriformes / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
keywords: amplicon sequencing; bioinformatics; copy number variation; next-generation sequencing; passerine MHC