Oakley, Brian B., Dowd, Scot E. and Purdy, Kevin J.. (2011) ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples. FEMS Microbiology Ecology, Vol.77 (No.1). pp. 17-27. ISSN 1574-6941

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Abstract

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples.

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Item Type:	Journal Article
Subjects:	Q Science > QA Mathematics > QA76 Electronic computers. Computer science. Computer software Q Science > QH Natural history > QH426 Genetics
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Polymerase chain reaction -- Computer programs, Biological assay -- Computer programs, Genetics -- Computer programs
Journal or Publication Title:	FEMS Microbiology Ecology
Publisher:	Wiley-Blackwell Publishing Ltd.
ISSN:	1574-6941
Date:	July 2011
Volume:	Vol.77
Number:	No.1
Page Range:	pp. 17-27
Identification Number:	10.1111/j.1574-6941.2011.01079.x
Status:	Peer Reviewed
Access rights to Published version:	Restricted or Subscription Access
Funder:	Sixth Framework Programme (European Commission) (FP6)
Grant number:	MEXT-CT-2005-024112 (FP6)
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URI:	http://wrap.warwick.ac.uk/id/eprint/37090

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