title: Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.
abstract: In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.
authors.affiliation: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Suchan
authors.affiliation: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
authors.forename: Camille
authors.initials: C
authors.lastname: Pitteloud
authors.affiliation: Biology Faculty, Lomonosov Moscow State University, Moscow, Russia.|InsideDNA Ltd., London, United Kingdom.
authors.forename: Nadezhda S
authors.initials: NS
authors.lastname: Gerasimova
authors.affiliation: InsideDNA Ltd., London, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Kostikova
authors.affiliation: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
authors.forename: Sarah
authors.initials: S
authors.lastname: Schmid
authors.affiliation: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
authors.forename: Nils
authors.initials: N
authors.lastname: Arrigo
authors.affiliation: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
authors.forename: Mila
authors.initials: M
authors.lastname: Pajkovic
authors.affiliation: Institute of Botany, Polish Academy of Sciences, Kraków, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Ronikier
authors.affiliation: Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.
authors.forename: Nadir
authors.initials: N
authors.lastname: Alvarez
mesh_terms: D000818:Animals; D002080:Butterflies / Q000235:genetics; D019295:Computational Biology; D004247:DNA / Q000235:genetics; D000068598:Data Accuracy; D015723:Gene Library; D056426:Genetic Loci; D023281:Genomics / Q000379:methods*; D006110:Grasshoppers / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013048:Specimen Handling
chemical_list: D004247:DNA