Additionally, the Escherichia coli position data was kindly provided by staff at the RDP. The downloaded sequences were filtered based on E. coli position. Only sequences with data present in the qPCR assay amplicon of interest were considered to be eligible for sequence matching for the particular qPCR assay. Numerical and taxonomic coverage analysis was performed for the BactQuant assay and a published qPCR assay [15] by developing a web service for the RDP Probe Match Tool for sequence matching. C. Overview of sequence matching analysis for determining assay coverage. All sequence matching for the in silico coverage analysis was performed using

two conditions: a) perfect match of full-length primer and probe sequences and b) perfect find more match of full-length probe sequence and the last 8 nucleotides of primer sequences at the 3´ end. For each sequence matching condition, the in silico coverage analysis was performed at three taxonomic levels: phylum, genus, and species, as well as for all sequences eligible for sequence Barasertib matching. The remaining taxonomic levels were omitted due to the large amounts of missing and inconsistent data. Details of in silico coverage analyses are as follows: D. Numerical coverage analysis. At each analysis level, unique operational taxonomic unit (OTU), i.e., each unique taxonomic group Ro 61-8048 research buy ranging from

unique phyla to unique species, containing at least one sequence that is a sequence match

(i.e., “match”) for all three components of the assay of interest were identified using the following requirement: [Forward Primer Perfect Match](union)[Reverse Primer Perfect Match](union)[Probe Perfect Match]. The in silico coverage analysis was performed in a stepwise fashion, beginning with all eligible sequences, then proceeding to analysis at the species-, genus-, and phylum-level. At each step, the taxonomic identification of each sequence was generated by concatenation of relevant taxonomic data (e.g., for species-level analysis, a unique taxonomic identification consisting of concatenated Phylum-Genus- species name was considered as one unique species). The sequence Exoribonuclease IDs were used in lieu of a taxonomic identification for the first analysis step, which included all eligible sequences. The stepwise numerical coverage analysis was performed as follows: all eligible sequences underwent sequence matching with all three components of the assays of interest using a select matching condition (i.e., the stringent or the relaxed criterion). The sequence IDs of matched sequences were assigned and binned as Assay Perfect Match sequence IDs. For this first analysis step, the numerical coverage was calculated using the total number of sequences with Assay Perfect Match sequence IDs as the numerator and the total number of eligible sequences as the denominator.