Wastewater Solids Nucleic Acid Extraction

Settled solids were obtained from 15 wastewater treatment plants across the United States (Table S2). Solids were collected from the primary clarifier or settled from a 24-h composited influent sample using Imhof cones. Samples were collected in sterile containers and transported to the lab. Samples from the Bay Area were processed immediately, while other samples were stored at −80°C until analysis (between 5 and 20 months). None of the samples stored at −80°C underwent a freeze-thaw cycle prior to the current work.
Solids were dewatered using centrifugation, and then an aliquot of the dewatered solids was set aside for dry-weight analysis. Solids were then suspended in a buffer (approximately 75 mg/mL), homogenized, and centrifuged. This suspension of solids in buffer was found to alleviate inhibition of RT-PCR (21 (link)). An aliquot of the supernatant was processed for total nucleic acid extraction using Chemagic 360 (Perkin Elmer). Nucleic acid preparations from wastewater samples are known to contain PCR inhibitors that interfere with their accurate quantification using PCR-based methods. Therefore, inhibitors were removed using the OneStep PCR inhibitor removal kit (Zymo Research; catalog no. D6035), yielding nucleic acids in 50 μL of eluant. These methods have been published in detail (42 (link)), and step-by-step protocols are available on protocols.io (43 , 44 ).