16S sequencing of slow sand filters
Amplicons of the V3  V4 region of the bacterial 16S rRNA gene were generated with universal bacterial primers Bact_341F (5-CCTACGGGNGGCWGCAG-3) and Bact_785R (5-GACTACHVGGGTATCTAATCC-3). Each PCR contained 12.2 l MQ water, 10 l 5PRIME HotMasterMix (Quantabio, USA), 0.8 l Bovine Serum Albumin (10 mg/ml), 0.5 l of both forward and reverse primers (10 M) and 1 l of DNA template. The PCR program started with 94C for 3 minutes and continued with 35 cycles of 94C for 45 s, 50C for 1 min and  72C for 1,5 min. After the cycling, there was a finishing step of 72C for 10 minutes.  Amplicons from each PCR reaction were quantified with the Qubit 2.0 dsDNA BR Assay Kit (Thermo Fisher Scientific, USA) and inspected visually with agarose gel electrophoresis. 50 ng of each replicate of amplicons (same distribution point and time of sampling) were pooled together and purified with UltraClean PCR Clean-up Kit (Qiagen, Germany) according to the manufacturers instructions. Sequence analysis was performed with a MiSeq Reagent Kit v3 (Illumina, USA) and run 600 cycles with 10 % PhiX according to the manufacturers instructions.