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Can long-range PCR be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics?: a case study within spiders (Arthropoda: Araneae)

Briscoe, Andrew G.; Goodacre, Sara; Masta, Susan E.; Taylor, Martin I.; Arnedo, Miquel A.; Penney, David; Kenny, John; Creer, Simon

Authors

Andrew G. Briscoe

Sara Goodacre

Susan E. Masta

Martin I. Taylor

Miquel A. Arnedo

David Penney

John Kenny

Simon Creer



Abstract

The development of second generation sequencing technology has resulted in the rapid production of large volumes of sequence data for relatively little cost, thereby substantially increasing the quantity of data available for phylogenetic studies. Despite these technological advances, assembling longer sequences, such as that of entire mitochondrial genomes, has not been straightforward. Existing studies have been limited to using only incomplete or nominally intra-specific datasets resulting in a bottleneck between mitogenome amplification and downstream high-throughput sequencing. Here we assess the effectiveness of a wide range of targeted long-range PCR strategies, encapsulating single and dual fragment primer design approaches to provide full mitogenomic coverage within the Araneae (Spiders). Despite extensive rounds of optimisation, full mitochondrial genome PCR amplifications were stochastic in most taxa, although 454 Roche sequencing confirmed the successful amplification of 10 mitochondrial genomes out of the 33 trialled species. The low success rates of amplification using long-Range PCR highlights the difficulties in consistently obtaining genomic amplifications using currently available DNA polymerases optimised for large genomic amplifications and suggests that there may be opportunities for the use of alternative amplification methods.

Journal Article Type Article
Publication Date May 8, 2013
Journal PLoS ONE
Electronic ISSN 1932-6203
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 8
Issue 5
Article Number 8
Institution Citation Briscoe, A. G., Goodacre, S., Masta, S. E., Taylor, M. I., Arnedo, M. A., Penney, D., …Creer, S. (2013). Can long-range PCR be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics?: a case study within spiders (Arthropoda: Araneae). PLoS ONE, 8(5), doi:10.1371/journal.pone.0062404
DOI https://doi.org/10.1371/journal.pone.0062404
Publisher URL http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0062404
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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