Last year, I profiled Suchan et al.’s “hyRAD” method for reduced-representation genome sequencing of degraded sources of DNA using RAD probes. While it’s too early to say whether hyRAD will be widely used by molecular ecologists looking to integrate historic samples into their work–in my own research, I found it effective, but not efficient–it remains a promising step forward for population genomics with a temporal dimension.
Now, the team behind hyRAD have published a major update to their protocol (“hyRAD-X”), replacing fragments from a RAD digest with messenger RNA as the basis for the probes in target capture. This, the authors argue, improves on the original method in several ways. First, as the reference used in bioinformatics steps is an assembled transcriptome from RNAseq of one high-quality tissue sample, both loci definition and SNP calling accuracy are improved. Second, the RNA probes in the method increase hybridization stringency — an important improvement for low-content DNA samples. (Arguably the main reason to apply hyRAD-X in the first place!)
So which method should you use? On the one hand, hyRAD-X’s advantages appear numerous, and address many of the problems inherent to the original protocol. Targeting the exome reduces the likelihood of capturing paralogous loci, as repetitive genomic regions are more frequently found in noncoding DNA. RNA probes are more thermodynamically stable, and eliminate the risk of contamination and chimera formation. Nor is hyRAD-X dramatically more complicated at the lab bench, deviating from hyRAD only in its probe generation step, where biotinylated RNA is synthesized from cDNA itself synthesized from a total RNA extract of a single high-quality sample. By integrating biotin-UTP during the in-vitro transcription stage of this process, the method also avoids the substantial probe material loss probably with the nick-labeling mechanisms proposed in Suchan et al. 2012. And in a comparison of the performance of both methods on an identical timeseries of conifer subfossils (spanning 7200 – 5800 years bp), Schmid et al. report significantly more SNPs from hyRAD-X than hyRAD with a de novo reference, and significantly greater mean read depth per site.
**Figure 2** from Schmid et al. 2017, diagramming the hyRAD-X method.
On the other hand, these benefits are not abundantly clear from the author’s published results. Schmid et al. report no differences in percentage of missing data, overall mean read depth, or chimera production. Concerningly, they do report 2-8 times higher PCR duplicates in hyRAD-X compared to hyRAD alignments. (I found PCR duplicates to a be pervasive feature of my own hyRAD captures, and are an important consideration when deciding sequencing effort.) And when comparing hyRAD-X and hyRAD data aligned to an identical transcriptome, the original hyRAD protocol resulted in a higher percentage of mapped reads and a greater SNPs, suggesting its primary limitations may have been related to the inherent problems with de novo reference genomes. Finally, putatively neutral loci may be preferable to exome data in some cases where sites under selection violate the assumptions of population genetic models, or bias phylogenetic inference.
However these issues come out in the wash, I think the decision to apply hyRAD, hyRAD-X, or another probe-based method for reduced representation sequencing historic samples is more likely to be based on your project’s goals and your lab’s contingencies than any clear advantages of a particular protocol. But while there may not yet be a silver bullet for population genomics with degraded DNA, it’s undoubtedly an exciting time to be working in the field, and hyRAD-X represents a creative solution to its many challenges.
References
Schmid, S., Genevest, R., Gobet, E., Suchan, T., Sperisen, C., Tinner, W., Alvarez, N. 2017. HyRAD-X, a versatile method combining exome capture and RAD sequencing to extract genomic information from ancient DNA. DOI: 10.1111/2041-210X.12785
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