title: Wastewater from treatment plant pipe
design: All DNA extractions were carried out using the Quick-DNA Miniprep kit (ZYMO Research, Irvine, CA)) according to the cell monolayer extraction protocol, with alterations to the cell lysis step for sample suitability. Lysis buffer was applied directly to samples in DNAse/RNAse free bags (VWR, Leicestershire, UK) and massaged to ensure complete coverage of the sample surfaces. Samples were left in lysis buffer for 10 min before the lysate was removed and processed in accordance with the manufacturers instructions. For the filtered water samples, the membranes were removed from the plastic Sterivex filter casing and DNA extracted as described above. Extracted DNA concentrations were measured using Qubit dsDNA High Sensitivity Assay Kit on a Qubit 4 Fluorometer (Thermo Scientific, Oxford, UK). The yields of DNA varied in the range 100-300 ng, which was sufficient for the 16S rRNA gene V4 amplicon sequencing. 16S rRNA gene amplicons were produced using duplicate amplification with double-indexed fusion primers as reported previously (Fadrosh et al., 2014). The hypervariable V4 16S rRNA gene region  was PCR-amplified with modified forward primer F515 (5-GTGBCAGCMGCCGCGGTAA-3) and reverse R806 prokaryotic primer (5-GGACTACHVGGGTWTCTAAT-3). Primers contained the Illumina adapters and sequencing primers, a 12 bp barcode sequence, a heterogeneity spacer, and 16S rRNA gene universal primers (Fadrosh et al., 2014). PCR was conducted using approx. 2 ng DNA template per reaction and  OneTaq DNA Polymerase reagents (New England Biolabs, Ipswich, MA, USA). All PCR batches were run with no-template negative control samples. PCR  conditions included: denaturation at 95 C for 2 min with following  30 cycles of denaturation at 95 C for 30 s, annealing at 50 C for 50 s, and DNA synthesis at 68 C for 30 s with a final elongation at 68 C for 5 min. PCR products  were visualised in a 1.2 %  (TAE) tris-acetate-EDTA agarose gel using a GelDoc System (Bio-Rad, Hercules, CA). DNA bands of approximately 440 bp were gel-purified using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). The purified amplicons were then quantified using the Qubit 4.0 Fluorometer, pooled in equimolar amounts and the final pool was run on Illumina MiSeq platform (Illumina Inc., San Diego, CA) using 500-cycle v2 chemistry (2  250 bp paired-end reads) at the Centre for Environmental Biotechnology, Bangor, United Kingdom.