Since RAD-seq was first developed, we’ve seen a number of new versions and approaches from an enthusiastic scientific community. The latest was recently published in PLoS One and demonstrates a RAD-based method suitable for analyzing degraded DNA, an essential step for studying samples stored in museum and other collections.
“Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens” comes from lead authors Tomasz Suchan and Camille Pitteloud at the University of Lausanne and their collaborators in Russia, Poland, and the UK. The project was launched to overcome the challenges of using traditional RAD-seq methods, which require longer DNA fragments than are typically available in museum samples. “Museum collections … have not necessarily ensured optimal conditions for DNA preservation,” the authors write. “As a result, many museum specimens yield highly fragmented DNA — even for relatively recently collected samples, limiting their use for molecular ecology, conservation genetics, phylogeographic and phylogenetic studies.”
Their solution is a method called hyRAD, for hybridization RAD, which starts by using double-digest RAD-seq to produce DNA fragments from fresh samples of the species of interest. Those fragments then become capture probes for use with the degraded DNA samples. “Our method thus combines the simplicity and relatively low cost of developing RAD-sequencing libraries with the power and accuracy of hybridization-capture methods,” the team reports. “This enables the effective use of low quality DNA and limits the problems caused by sequence polymorphisms at the restriction site.”
The scientists tested this protocol on eight samples of Lycaena helle butterflies, followed by a validation project on 49 samples of the Palearctic grasshopper Oedaleus decorus. Like other RAD methods, they used Pippin Prep for size selection prior to sequencing.
“Not relying on the presence of restriction site, the method presented here should be also useful for broader phylogenetic scales, allowing sequencing homologous loci from more divergent taxa, which would not be possible to retrieve using classical RAD-seq approaches,” the scientists conclude.