16S- subsea tunnel concrete biofilm metagenomic data
The V4 hyper-variable region of the 16S-rRNA gene was amplified in duplicates by Biometra T3000 Thermocycler using the primers 515F and 806R to cover both bacteria and archaea (Caporaso et al. 2011; Hugerth et al. 2014), with dual-index labelling (Kozich et al. 2013). The following PCR program was used: Activation at 98C for 30 sec, denaturation at 98C for 10 sec, annealing at 55.8C for 30 sec, and extension at 72C for 30 sec for 30 cycles, followed by final elongation at 72C for 10 min. The PCR reaction (20 l) contained Phusion Hot Start II DNA Polymerase (0.2 l) (Thermo Scientific), 5X Phusion HF Buffer (4 l), 10 mM dNTPs (0.4 l), DMSO (0.6 l), template DNA (1 l) and primers (10 M, 1 l each). All PCR reactions were run in duplicates. DNA concentrations of purified samples were measured with a Qubit 2.0 fluorometer (Life Technologies), normalized at 6 nM and analysed with Miseq Illumina sequencer.