Can long-range PCR be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics?: a case study within spiders (Arthropoda: Araneae)
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 lon...
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Public Library of Science
2013
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| Online Access: | https://eprints.nottingham.ac.uk/2802/ |
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| author | Briscoe, Andrew G. Goodacre, Sara Masta, Susan E. Taylor, Martin I. Arnedo, Miquel A. Penney, David Kenny, John Creer, Simon |
| author_facet | Briscoe, Andrew G. Goodacre, Sara Masta, Susan E. Taylor, Martin I. Arnedo, Miquel A. Penney, David Kenny, John Creer, Simon |
| author_sort | Briscoe, Andrew G. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T18:19:38Z |
| format | Article |
| id | nottingham-2802 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T18:19:38Z |
| publishDate | 2013 |
| publisher | Public Library of Science |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-28022020-05-04T16:37:00Z https://eprints.nottingham.ac.uk/2802/ 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 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. Public Library of Science 2013-05-08 Article PeerReviewed Briscoe, Andrew G., Goodacre, Sara, Masta, Susan E., Taylor, Martin I., Arnedo, Miquel A., Penney, David, 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 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0062404 doi:10.1371/journal.pone.0062404 doi:10.1371/journal.pone.0062404 |
| spellingShingle | Briscoe, Andrew G. Goodacre, Sara Masta, Susan E. Taylor, Martin I. Arnedo, Miquel A. Penney, David Kenny, John Creer, Simon Can long-range PCR be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics?: a case study within spiders (Arthropoda: Araneae) |
| title | Can long-range PCR be used to amplify genetically
divergent mitochondrial genomes for comparative
phylogenetics?: a case study within spiders (Arthropoda:
Araneae) |
| title_full | Can long-range PCR be used to amplify genetically
divergent mitochondrial genomes for comparative
phylogenetics?: a case study within spiders (Arthropoda:
Araneae) |
| title_fullStr | Can long-range PCR be used to amplify genetically
divergent mitochondrial genomes for comparative
phylogenetics?: a case study within spiders (Arthropoda:
Araneae) |
| title_full_unstemmed | Can long-range PCR be used to amplify genetically
divergent mitochondrial genomes for comparative
phylogenetics?: a case study within spiders (Arthropoda:
Araneae) |
| title_short | Can long-range PCR be used to amplify genetically
divergent mitochondrial genomes for comparative
phylogenetics?: a case study within spiders (Arthropoda:
Araneae) |
| title_sort | can long-range pcr be used to amplify genetically
divergent mitochondrial genomes for comparative
phylogenetics?: a case study within spiders (arthropoda:
araneae) |
| url | https://eprints.nottingham.ac.uk/2802/ https://eprints.nottingham.ac.uk/2802/ https://eprints.nottingham.ac.uk/2802/ |