Abstract
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.
Highlights
The analysis of the microbial community structure and abundance based on universal conserved marker genes has become a powerful tool for many disciplines in life science with a specific focus on next-generation sequencing technologies[1,2]
Genetic markers commonly used for microarrays and quantitative polymerase chain reaction (qPCR) are ribosomal RNA genes, e.g. 16S, for bacterial communities[3,4], as well as functional genes that determine microbial community structure with regards to specific metabolic functions[5,6]
Both technologies rely on taxon-specific short nucleotide sequences of the marker gene of interest, so-called oligonucleotide probes (OPs)
Summary
The analysis of the microbial community structure and abundance based on universal conserved marker genes has become a powerful tool for many disciplines in life science with a specific focus on next-generation sequencing technologies[1,2] In addition to these qualitative methods technologies such as environmental microarrays and quantitative polymerase chain reaction (qPCR) offer cost-effective and highly reproducible techniques for semiquantitative estimation of microbial communities. Genetic markers commonly used for microarrays and qPCR are ribosomal RNA (rRNA) genes, e.g. 16S, for bacterial communities[3,4], as well as functional genes that determine microbial community structure with regards to specific metabolic functions[5,6] Both technologies rely on taxon-specific short nucleotide sequences of the marker gene of interest, so-called oligonucleotide probes (OPs). ProbeSpec is the only batch probe specificity assessment tool which provides interactive manipulation of specificity and sensitivity thresholds
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