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. and Goodacre, Sara and Masta, Susan E. and Taylor, Martin I. and Arnedo, Miquel A. and Penney, David and Kenny, John and Creer, Simon (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). 8/1-8/8. ISSN 1932-6203

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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.

Item Type: Article
Schools/Departments: University of Nottingham UK Campus > Faculty of Medicine and Health Sciences > School of Life Sciences
Identification Number: https://doi.org/10.1371/journal.pone.0062404
Depositing User: Davies, Mrs Sarah
Date Deposited: 23 Apr 2014 10:55
Last Modified: 13 Sep 2016 16:45
URI: http://eprints.nottingham.ac.uk/id/eprint/2802

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