Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes

PLoS One. 2019 Feb 4;14(2):e0209499. doi: 10.1371/journal.pone.0209499. eCollection 2019.

Abstract

Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make "phylogenetically diverse" probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

Grants and funding

Laboratory work was funded by the following grants: CF14-0461: Recovery of lost genetic diversity in barley awarded to BLM from the Carlsberg Foundation; LP130100648: Identifying the diversity and evolution of loci associated with adaptation to aridity/heat and salinity in ancient cereal crops awarded to MG and AC from the Australian Research Council. Natural Trap Cave excavations were funded by U.S. NSF EAR/SGP# 1425059 awarded to JM and AC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.