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Type: Journal article
Title: Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes
Author: Richards, S.
Hovhannisyan, N.
Gilliham, M.
Ingram, J.
Skadhauge, B.
Heiniger, H.
Llamas, B.
Mitchell, K.
Meachen, J.
Fincher, G.
Austin, J.
Cooper, A.
Citation: PLoS ONE, 2019; 14(2):e0209499-1-e0209499-18
Publisher: Public Library Science
Issue Date: 2019
ISSN: 1932-6203
Statement of
Stephen M. Richards, Nelli Hovhannisyan, Matthew Gilliham, Joshua Ingram, Birgitte Skadhauge, Holly Heiniger, Bastien Llamas, Kieren J. Mitchell, Julie Meachen, Geoffrey B. Fincher, Jeremy J. Austin, Alan Cooper
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.
Keywords: Bison; mitochondrial DNA; probe hybridization; marsupials; mitochondria; DNA libraries; millet; animal phylogenetics
Rights: © 2019 Richards et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RMID: 0030108159
DOI: 10.1371/journal.pone.0209499
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