title: Double-digest RAD sequencing using Ion Proton semiconductor platform (ddRADseq-ion) with nonmodel organisms.
abstract: Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double-digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single-end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11,000 polymorphic loci per library of 6-30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost-effective generation of variable and reproducible genetic markers.
authors: Institute of Biodiversity, Animal Health & Comparative Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
authors: Hans
authors: H
authors: Recknagel
authors: Institute of Biodiversity, Animal Health & Comparative Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
authors: Arne
authors: A
authors: Jacobs
authors: Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, University of Glasgow, Garscube Campus, Bearsden, G61 1QH, UK.|Institute of Molecular, Cell & Systems Biology, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
authors: Pawel
authors: P
authors: Herzyk
authors: Institute of Biodiversity, Animal Health & Comparative Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
authors: Kathryn R
authors: KR
authors: Elmer
mesh_terms: D000818:Animals; D019295:Computational Biology / Q000379:methods; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008116:Lizards / Q000145:classification / Q000235:genetics*; D010802:Phylogeny; D011110:Polymorphism, Genetic; D012487:Salmonidae / Q000145:classification / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D014337:Trout / Q000145:classification / Q000235:genetics*
chemical_list: D004262:DNA Restriction Enzymes
keywords: Ion Proton; Ion Torrent; RAD sequencing; double-digest; genotyping by sequencing; semiconductor sequencing; single nucleotide polymorphism (SNP) genotyping