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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Autores principales:	Andrew G Briscoe, Sara Goodacre, Susan E Masta, Martin I Taylor, Miquel A Arnedo, David Penney, John Kenny, Simon Creer
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a5662f93ee984f21a7f90d23ec2897cb
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spelling	oai:doaj.org-article:a5662f93ee984f21a7f90d23ec2897cb2021-11-18T07:46:22ZCan long-range PCR be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics? A case study within spiders (Arthropoda: Araneae).1932-620310.1371/journal.pone.0062404https://doaj.org/article/a5662f93ee984f21a7f90d23ec2897cb2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23667474/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Andrew G BriscoeSara GoodacreSusan E MastaMartin I TaylorMiquel A ArnedoDavid PenneyJohn KennySimon CreerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e62404 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Andrew G Briscoe Sara Goodacre Susan E Masta Martin I Taylor Miquel A Arnedo David Penney John Kenny Simon Creer Can long-range PCR be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics? A case study within spiders (Arthropoda: Araneae).
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.
format	article
author	Andrew G Briscoe Sara Goodacre Susan E Masta Martin I Taylor Miquel A Arnedo David Penney John Kenny Simon Creer
author_facet	Andrew G Briscoe Sara Goodacre Susan E Masta Martin I Taylor Miquel A Arnedo David Penney John Kenny Simon Creer
author_sort	Andrew G Briscoe
title	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_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_sort	can long-range pcr be used to amplify genetically divergent mitochondrial genomes for comparative phylogenetics? a case study within spiders (arthropoda: araneae).
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/a5662f93ee984f21a7f90d23ec2897cb
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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).

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