Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens.

Tomasz Suchan,Michał Ronikier,Anna Kostikova,Nadir Alvarez,Sarah Schmid,Nadezhda S Gerasimova,Camille Pitteloud,Mila Pajkovic,Nils Arrigo,Ludovic Orlando

doi:10.1371/journal.pone.0151651

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.

Highlights

With the advent of next-generation sequencing, conducting genomic-scale studies on nonmodel species has become a reality [1]
The cost of genome sequencing has substantially dropped over the last decade and repositories encompass an incredible amount of genomic data, which has opened avenues for the emerging field of ecological genomics
The wide utility of RAD-sequencing in ecological, phylogenetic and phylogeographic studies is limited by two main factors: i) the quality of the starting genomic DNA; ii) the degree of divergence among the studied specimens, that translates into DNA sequence polymorphism at the restriction sites targeted by the RAD protocols

Summary

Introduction

With the advent of next-generation sequencing, conducting genomic-scale studies on nonmodel species has become a reality [1]. Among the most popular are approaches relying on hybridization capture of exome [4] or conserved fragments of the genome [5], RNA sequencing (RNAseq [6]), and Restriction-Associated-DNA sequencing (RADseq [7, 8]). The latter has been developed in many different versions, but generally relies on specific enzymatic digestion and further selection of a range of DNA fragment sizes. The wide utility of RAD-sequencing in ecological, phylogenetic and phylogeographic studies is limited by two main factors: i) the quality of the starting genomic DNA; ii) the degree of divergence among the studied specimens, that translates into DNA sequence polymorphism at the restriction sites targeted by the RAD protocols

Methods

Results

Conclusion