title: Less is more: extreme genome complexity reduction with ddRAD using Ion Torrent semiconductor technology.
abstract: Massively parallel sequencing a small proportion of the whole genome at high coverage enables answering a wide range of questions from molecular evolution and evolutionary biology to animal and plant breeding and forensics. In this study, we describe the development of restriction-site associated DNA (RAD) sequencing approach for Ion Torrent PGM platform. Our protocol results in extreme genome complexity reduction using two rare-cutting restriction enzymes and strict size selection of the library allowing sequencing of a relatively small number of genomic fragments with high sequencing depth. We applied this approach to a common freshwater fish species, the Eurasian perch (Perca fluviatilis L.), and generated over 2.2 MB of novel sequence data consisting of ~17,000 contigs, identified 1259 single nucleotide polymorphisms (SNPs). We also estimated genetic differentiation between the DNA pools from freshwater (Lake Peipus) and brackish water (the Baltic Sea) populations and identified SNPs with the strongest signal of differentiation that could be used for robust individual assignment in the future. This work represents an important step towards developing genomic resources and genetic tools for the Eurasian perch. We expect that our ddRAD sequencing protocol for semiconductor sequencing technology will be useful alternative for currently available RAD protocols.
authors.affiliation: Department of Aquaculture, Estonian University of Life Sciences, Tartu, 51006, Estonia.
authors.forename: Lilian
authors.initials: L
authors.lastname: Pukk
authors.affiliation: Department of Information Technology, University of Turku, Turku, 20014, Finland.
authors.forename: Freed
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: Bioinformatics Lab, QIMR Berghofer Medical Research Institute, Brisbane, Qld, Australia.|School of Medicine, The University of Queensland (UQ), Brisbane, Qld, 4006, Australia.
authors.forename: Shihab
authors.initials: S
authors.lastname: Hasan
authors.affiliation: Institute of Technology, University of Tartu, Tartu, 50411, Estonia.
authors.forename: Veljo
authors.initials: V
authors.lastname: Kisand
authors.affiliation: Department of Aquaculture, Estonian University of Life Sciences, Tartu, 51006, Estonia.
authors.forename: Riho
authors.initials: R
authors.lastname: Gross
authors.affiliation: Department of Biology, University of Turku, Turku, 20520, Finland.
authors.forename: Anti
authors.initials: A
authors.lastname: Vasemägi
mesh_terms: D000818:Animals; D014644:Genetic Variation; D005828:Genetics, Population / Q000379:methods; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010471:Perches / Q000145:classification / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: DNA pooling; Eurasian perch (Perca fluviatilis L.); SNP development and validation; de novo assembly; next-generation sequencing; population genomics