16S rRNA sequencing of cathodic and anodic biofilm from a BES for PCE removal
The extracted DNA was employed for 16S rRNA gene amplicon sequencing, targeting the V1-V3 region of bacterial 16S rRNA (Matturro et al., 2017). PCR reactions were performed in duplicate, in 25L total volume, including 4 ng of DNA template, 0.5M of the library adaptors with V13 primers (27F: 5-AGAGTTTGATCCTGGCTCAG-3; 534R: 5-ATTACCGCGGCTGCTGG-3) and 1x Phusion Flash High Fidelity Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The libraries were purified using the Agencourt AMpure XP bead, according to the protocol instructions (Beckmann Coulter, Italy). Library concentration was determined with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA USA). The purified libraries were pooled in equimolar concentrations and diluted to 4nM. In order to overcome low complexity matter, 20 % Phix control was added to the library. The samples were paired end sequenced (2301 bp) on a MiSeq platform (Illumina, San Diego, CA, USA) using a MiSeq Reagent kit v3, 600 cycles (Illumina) following the standard procedures for preparing and loading samples.