title: 16s RNA-Seq of freshwater metagenome
design: We filtered between 1 L and 2.5 L of water, depending on turbidity levels, on to a 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. We sequenced the prokaryotic 16S ribosomal RNA gene to characterize the bacterial community in surface water and fecal samples. Polymerase chain reaction (PCR) and amplicon sequencing was performed by Novogene Corporation Inc. The V3-V4 region of the 16S rRNA gene region was amplified using dual indexed primers 341F (5 CCTAYGGGRBGCASCAG- 3) and 806R (5-GGACTACNNGGGTATCTAAT-3) (35). 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. Novogene verified library quality, quantity, and amplicon length with Qubit and real-time PCR, at which point libraries were pooled using Illuminas NEBNext UltraTM DNA Library Prep Kit.