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ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples
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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. doi:10.1111/j.1574-6941.2011.01079.x
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Official URL: http://dx.doi.org/10.1111/j.1574-6941.2011.01079.x
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.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QA Mathematics > QA76 Electronic computers. Computer science. Computer software Q Science > QH Natural history > QH426 Genetics |
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| 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 | ||||
| Official Date: | July 2011 | ||||
| Dates: |
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| Volume: | Vol.77 | ||||
| Number: | No.1 | ||||
| Page Range: | pp. 17-27 | ||||
| DOI: | 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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