16s RNA-Seq of wastewater metagenome: Jackson wastewater influent
Wastewater influent samples (volumes ranging from 100-200 ml) were collected throughout the duration of the sampling season from two wastewater treatment facilities in the study area (Town of Jackson and Aspen Pines wastewater treatment plants). These samples were filtered onto 0.45 m sterile nitrocellulose filter (Pall Corporation, Port Washington, NY, U.S.A). We extracted the total DNA from the filters using the Qiagen DNEasy PowerWater (Qiagen, Hilden, Germany) kit according to manufacturers instruction. DNA sample quality and quantity were measured via NanoDrop spectrophotometer (Fisher Scientific, Waltham, MA, U.S.A) prior to sequencing. Polymerase chain reaction (PCR) and amplicon sequencing was performed by Novogene Corporation Inc. Novogene sequenced the V3-V4 region of the prokaryotic 16S ribosomal RNA gene using dual indexed primers 341F (5 CCTAYGGGRBGCASCAG- 3) and 806R (5-GGACTACNNGGGTATCTAAT-3). All PCR reactions were performed with the Phusion  High-Fidelity Master Mix (New England Biolabs). Novogene used gel electrophoresis to select PCR products of adequate length for sequencing and were purified using Qiagen Gel Extraction Kit. Equal quantities of PCR product from each sample were pooled, end-repaired, A-tailed, and further ligated with Illumina adapters to create sequencing libraries. Library quality, quantity, and amplicon length was verified with Qubit and real-time PCR, at which point libraries were pooled using Illuminas NEBNext UltraTM DNA Library Prep Kit. Novogene performed quality filtering to obtain high-quality clean sequence reads and removed primers and adapters before returning FASTA files.