16S rRNA amplicon sequencing of blank sample
Filtering of water samples and extraction of DNA using the standard protocol for Dneasy PowerWater Kit (Qiagen, Germany). Duplicate amplification of the 16S rRNA gene variable region 4 (abV4C): [515FB] GTGYCAGCMGCCGCGGTAA and [806RB] GGACTACNVGGGTWTCTAAT. PCRwas done with the following program: Initial denaturation at 95 C for 2 min, 30 cycles of amplification (95 C for 15 s, 55 C for 15 s, 72 C for 50 s) and a final elongation at 72 C for 5 min. The purified sequencing libraries were pooled in equimolar concentrations and diluted to 2 nM. The samples were pairedend sequenced (2x300 bp) on a MiSeq (Illumina, USA) using a MiSeq Reagent kit v3 (Illumina, USA) following the standard guidelines for preparing and loading samples on the MiSeq. > 10 % PhiX control library was spiked in to overcome low complexity issues often observed with amplicon samples.