DNA metabarcoding of biofilm
The 16S rRNA region was amplified using the primers Bakt 341F (5 CCT ACG GGN GGC WGC AG 3) and Bakt 805R (5 GAC TAC HVG GGT ATC TAA TCC 3) (Herlemann et al. 2011), to which the Illumina sequencing primer sequences were attached to their 5 ends. PCRs were carried out in a final volume of 25 L, containing 1 L of genomic DNA, 0.5 M of each forward and reverse primer, 12.5 L of Supreme NZYTaq 2x Green Master Mix (NZYTech, Lisbon, Portugal), and ultrapure water up to 25 L. The reaction mixture was incubated as follows: initial denaturation at 95 C for 5 min, followed by 25 cycles of 95 C for 30 s, 50 C for 30 s, 72 C for 30 s, and a final extension step at 72 C for 10 minutes. Samples were individually tagged in the second PCR round with identical conditions but only 5 cycles and 60 C as the annealing temperature. Libraries were purified using the Mag-Bind RXNPure Plus magnetic beads (Omega Biotek, Norcross, GA, USA), following the instructions provided by the manufacturer.