While the RAPTURE may have arrived, the development of novel restriction digest-based library prepartation techniques — and portmanteaus — continues unabated. In a paper published in PLoS ONE last month (and previously available as a preprint on bioRxiv), Tomasz Suchan and colleagues describe the latest of these methods, introducing “hyRAD” to the molecular ecology lexicon.
hyRAD (from hybridization RAD sequencing) differs from its predecessors in employing fragments generated from a standard double-digest RAD protocol as biotinylated probes. These probes are then hybridized with fragments from shotgun genomic libraries, capturing homologous sequences for PCR enrichment. By sequencing both the original ddRAD library and the enriched shotgun library, short reads from the latter can be assembled into contigs and mapped onto the former. (Because its protocol differs significantly from other RADseq variants, hyRAD requires a unique bioinformatic pipeline, which is also presented in the paper.)
The approach offers two distinct and related advantages. First, because hyRAD doesn’t rely on the presence of restriction sites, it improves among-sample loci coverage, reducing concerns of allelic dropout that plague RADseq seminar discussions everywhere. Second, this same attribute makes hyRAD uniquely adapted for use on degraded or historical DNA sequences, such as those extracted from museum specimens. Suchan and coauthors demonstrate its efficacy on museum and fresh tissue samples of 1) a non-model butterfly (Lycaena helle), and 2) the grasshopper Oedaleus decorus. In both examples, an impressive 70-80% of reads from museum samples were retained subsequent to quality filtering steps at the processing stage.
Is hyRAD right for you? It comes down to evolutionary timescale and budget. Previous phylogenomic studies using DNA from museum specimens have employed either sequence capture or whole genome shotgun sequencing. While sequence capture of the short “core” UCE region is effective for more distantly related taxa, extending to the flanking regions of UCEs — the sequences that contain a high proportion of variant sites and thus make the marker relevant for population-level timescales — requires longer fragments than the 100-200 bp typical of highly degraded DNA. Traditional sequence capture also requires prior knowledge of your organism for probe design, which is costly and time consuming. Similarly, shotgun approaches remain expensive and impractical for the large number of samples typical of population genetics and phylogeography.
In contrast, hyRAD builds off the known strength of RADseq for shallow timescales, and remains relatively cheap and simple. I suspect it will be used widely in the years to come.
Suchan, T., Pitteloud, C., Gerasimova, N.S., Kostikova, A., Schmid, S., Arrigo, N., Pajkovic, M., Ronikier, M., Alvarez, N. 2016. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens. PLoS One. DOI: 10.1371/journal.pone.0151651
McCormack, J., Tsai, W.L.E., Faircloth, B.C. 2015. Sequence capture of ultraconserved elements from bird museum specimens. Molecular Ecology Resources. DOI: 10.1111/1755-0998.12466
Besnard, G., Bertrand, J.A.M., Delahie, B., Bourgeois, Y.X.C., Lhuillier, E., Thebaud, C. 2016. Valuing museum specimens: high throughput DNA sequencing on historical collections of New Guinea crowned pigeons (Goura). Biological Journal of the Linnean Society. DOI: 10.1111/bij.12494
Ali, O. A., O’Rourke, S. M., Amish, S. J., Meek, M. H., Luikart, G., Jeffres, C., Miller, M. R. 2015. RAD Capture (Rapture): Flexible and efficient sequence-based genotyping. Genetics. DOI: 10.1534/genetics.115.183665