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Richards SM, Hovhannisyan N, Gilliham M, et al. Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes. PLoS One. 2019;14(2):e0209499doi: 10.1371/journal.pone.0209499.
Richards, S. M., Hovhannisyan, N., Gilliham, M., Ingram, J., Skadhauge, B., Heiniger, H., Llamas, B., Mitchell, K. J., Meachen, J., Fincher, G. B., Austin, J. J., & Cooper, A. (2019). Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes. PloS one, 14(2), e0209499. https://doi.org/10.1371/journal.pone.0209499
Richards, Stephen M, et al. "Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes." PloS one vol. 14,2 (2019): e0209499. doi: https://doi.org/10.1371/journal.pone.0209499
Richards SM, Hovhannisyan N, Gilliham M, Ingram J, Skadhauge B, Heiniger H, Llamas B, Mitchell KJ, Meachen J, Fincher GB, Austin JJ, Cooper A. Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes. PLoS One. 2019 Feb 04;14(2):e0209499. doi: 10.1371/journal.pone.0209499. eCollection 2019. PMID: 30716066; PMCID: PMC6361428.
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PLoS One. 2019 Feb 04;14(2):e0209499. doi: 10.1371/journal.pone.0209499. eCollection 2019.

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

PloS one

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

Affiliations

  1. Australian Centre for Ancient DNA, University of Adelaide, Adelaide, Australia.
  2. Yerevan State University, Yerevan, Armenia.
  3. ARC Centre of Excellence in Plant Energy Biology, Waite Research Institute, University of Adelaide, Adelaide, Australia.
  4. Carlsberg Research Laboratory, Copenhagen, Denmark.
  5. Des Moines University, Des Moines, Iowa, United States of America.
  6. ARC Centre of Excellence in Cell Walls, Waite Research Institute, University of Adelaide, Adelaide, Australia.

PMID: 30716066 PMCID: PMC6361428 DOI: 10.1371/journal.pone.0209499

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

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