Quantifying Analytical Sensitivity of CFU Titrations within the Microbiome of a Freshwater Aquarium
Titrated colony forming units (10^6 CFU to 10^1 CFU) of three Gram-negative bacteria: E. coli, S. marcescens, K. pneumoniae, and two Gram-positive bacteria: S. aureus and E. faecalis were spiked into 500mL of fresh water, filtered with borosilicate glass filtration (GF/F) filters, extracted with RNeasy PowerWater kit (Qiagen), reverse-transcribed with Superscript IV (Invitrogen) and sequenced with unbiased 16s, 18s and ITS shotgun sequencing, alongside a background H2O (0) control. Triplicate replicate extractions were pooled into a single sample for sequencing.