Performance and automation of ancient DNA capture with RNA hyRAD probes

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordData Availability: The datasets generated for this study can be accessed in the ENA (Accession Number PRJEB43744). The program for piloting the Opentrons OT-2 robot can be accessed at https://github.com/TomaszSuchan/opentrons-hyRAD.DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on pre-selected genomic regions. This feature is especially useful when analyzing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve as baits for capturing homologous fragments from aDNA libraries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing extremely low endogenous content (20-30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensitive to the original endogenous DNA content. Procedures based on two, instead of a single round of capture, increased on-target coverage up to 3.6-fold. Additionally, we used methylation sensitive restriction enzymes to produce probes targeting hypomethylated regions, which improved data quality by reducing post-mortem DNA damage and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol leveraging inexpensive robotic platforms to facilitate capture processing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples.University Paul Sabatier IDEX Chaire d’ExcellenceCNRSEuropean Union Horizon 2020Russian Foundation for Basic Researc