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High-throughput molecular methods such as quantitative polymerase chain reaction (qPCR) and environmental microarrays are cost-effective methods for semi-quantitative assessment of bacterial community structure and the identification of specific target organisms. Both techniques rely on short nucleotide sequences, so-called oligonucleotide probes, which require high specificity to the organisms in question to avoid cross-hybridization with non-target taxa. However, designing oligonucleotide probes for novel taxa or marker genes that show sufficient phylogenetic sensitivity and specificity is often time- and labor-intensive, as each probe has to be in-silico tested for its specificity and sensitivity. Here we present ProbeSpec, to our knowledge the first batch sensitivity and specificity estimation and visualization tool for oligonucleotide probes integrated into the widely used ARB software. Using ProbeSpec’s interactive “mismatch threshold” and “clade marked threshold” we were able to reduce the development time of highly specific probes for a recently published environmental oligonucleotide microarray from several months to one week.

ProbeSpec: batch specificity testing and visualization of oligonucleotide probe sets implemented in ARB