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ThermoPhyl is a free software tool that identifies Thermodynamically and Phylogenetically optimal assays for FISH, traditional PCR or TaqMan quantitative-PCR.

The problem:

Specifically and sensitively targeting a genotype of interest is critical for the success of many PCR-based queries of environmental or clinical samples containing multiple DNA or RNA templates. Recent next-generation sequencing efforts have shown that environmental samples may routinely contain genomes from hundreds to tens of thousands of operational taxonomic units (OTUs); a level of richness which clearly precludes the possibility of evaluating the specificity and sensitivity of candidate assays based on manual assessments of multiple sequence alignments.

A solution:

As a solution to this problem we have developed and validated a free software tool that is designed to harness the power of large sequence datasets now routinely available. ThermoPhyl identifies optimal assays by assessing a large number of candidate assays for phylogenetic sensitivity and specificity by comparison to a dataset of user-defined target and non-target sequences. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and is suitable for applications using one probe (e.g. FISH), two primers (traditional PCR), or two primers with an internal probe (e.g. TaqMan qPCR).


If you use ThermoPhyl we would appreciate it if you reference the following paper:

Oakley BB, Dowd SE, and Purdy KJ. (2011) ThermoPhyl: a software tool for designing thermodynamically and phylogenetically optimized quantitative-PCR assays. FEMS Microbiology Ecology 77: 17-27.

This paper can be downloaded from


Download ThermoPhyl (Perl can be downloaded from PERL)