3, the triplicates were compared and if a clear outlier was present (ΔCt > 0.3 from other two replicates), the outlier well was excluded from analysis. Amplification profiles of the seven selleck conditions tested were annotated and presented in Figure2A-B and Additional file 4: Figure S 4A-E. Results from laboratory quantitative validation using all conditions tested were summarized AUY-922 clinical trial in Table4. Detailed results of inter- and intra-run

coefficient of variation for Ct value and copy number were presented for all conditions tested in Figure3 and Additional file 5: Supplemental file 1A-C using scattered plots generated with the vegan package in R [18, 19]. Figure 2 A-B. Standard curve amplification profiles of the BactQuant assay generated from 10 μl and 5 μl reactions using seven ten-fold dilutions and normalized plasmid standards at 10 9 copies/μl. The Ct value of standard curve using 5 μl reaction volumes (Figure2B) shows an approximately 1 Ct left shift from the 10 μl reaction volumes https://www.selleckchem.com/products/tideglusib.html (Figure2A). However, the overall amplification profiles are not significantly different between the different reaction volumes over the assay dynamic range of 102 copies to 108 copies of 16 S rRNA gene per reaction. Table 4 Laboratory quantitative validation results of the BactQuant assay performed using pure plasmid standards and different mixed templates Templates used Assay dynamic range Average

reaction efficiency (SD) r 2 –value Plasmid standards–only (10 μl Rxn) 100–108 copies 102% (2%) >0.999 Plasmid standards-only (5 μl Rxn) 100 – 108 copies 95% (1%) >0.999 Plasmid standards plus 0.5 ng human gDNA 100 – 108 copies 99% (4%) >0.994 Plasmid standards plus 1 ng human gDNA 100 – 108 copies 101% (5%) >0.994 PIK3C2G Plasmid standards plus 5 ng human gDNA 500 – 108 copies 96% (1%) >0.999 Plasmid standards plus 10 ng human gDNA 1000 – 108 copies 97% (2%) >0.999 Plasmid standards plus 0.5 ng  C. albicans gDNA 100 – 108 copies 97% (1%) >0.999

Figure 3 Inter- and intra-run coefficient of variation (CoV) for 10 μl and 5 μl reactions using seven ten-fold dilutions and normalized plasmid standards at 10 9 copies/μl calculated using data from multiple runs. The data is presented for both copy number ( solid line) and Ct value ( dashed line). As would be expected, the CoV is higher for copy number than for Ct value and is also higher for inter-run than for intra-run. The CoV for copy number for both reaction volumes was consistently below 15% until at 107 copies for 5 μl reactions. The CoV for Ct value was consistently below 5% for both reaction volumes. Bacteria-to-human ratio calculations Calculations were performed using the following copy number and genome size estimates: the average bacterial 16 S rRNA gene copy number per genome was estimated to be 3.94 copies as calculated by rrnDB [20] (accessed at http://​ribosome.​mmg.​msu.​edu/​rrndb/​index.​php) and the average human 18 S rRNA gene copy number per genome was estimated to be 400 copies [21].