All wastewater samples (n = 168) were filtered in triplicate through sterile polycarbonate membranes (0.22-μm porosity; Whatman, UK) and stored at −80ºC until DNA extraction. Volumes of 25 to 50 ml of influent wastewater samples or of 150 to 400 ml of final effluent wastewater samples were filtered. Total DNA was extracted in triplicate, at once for the same sampling campaign, using the PowerWater DNA Isolation Kit (MO BIO Laboratories Inc., CA, USA) according to the manufacturer’s instructions, and quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA). For each sampling date, a DNA pool from the triplicates was prepared, resulting in one DNA extract per day and UWTP and three extracts per sampling campaign. DNA extract pools were shipped to the Michigan State University and run using the qPCR array, as previously described (24). Each partner kept an aliquot of the DNA pools for additional analyses as needed.

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