Open AccessThermoPhyl: a software tool for selecting phylogenetically optimized conventional and quantitative‐PCR taxon‐targeted assays for use with complex samples
Author(s) -
Oakley Brian B.,
Dowd Scot E.,
Purdy Kevin J.
Publication year - 2011
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.2011.01079.x
Subject(s) - biology , taqman , computational biology , phylogenetic tree , primer (cosmetics) , pyrosequencing , oligonucleotide , polymerase chain reaction , genetics , gene , chemistry , organic chemistry
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 that 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 that automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user‐defined target and nontarget sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimized candidate assays derived from primer design software packages. ThermoPhyl derives its name from its central premise of testing Thermo dynamically optimal assays for Phyl ogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan ® qPCR) application 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.