16S rRNA pyrosequences from hydrothermal vent fluids/Juan de Fuca Ridge
(4–12ng) were PCR-amplified using Takara Ex taq premix (Fisher). All PCR reactions were performed in a final volume of 50µL containing 25µL of Premix Taq, 1 µM of each primer and sterile MiliQ H2O to up to 50 µL. PCR amplifications were performed targetting the V1-V3 region of the 16S rRNA gene, using a general reverse primer (R519: 5'-CAGGWATTACCGCGGCKGCTG) combined with B primer (Roche: CCTATCCCCTGTGTGCCTTGGCAGTCT) in combination with unique tagged forward primer (F63-targeted: 5'-XXXXXXXXXXCAGGCCTAACACATGCAAGTC, where X represents different tags (64 in total)) combined with A primer (Roche: CCATCTCATCCCTGCGTGTCTCCGACTCAG). PCR conditions were as follows: after a denaturing step of 30s at 98°C, samples were processed through 30 cycles consisting of 10s at 98°C, 30s at 55°C and 30s at 72°C. The final extension step was done at 72°C for 4m30s. Following amplification, samples were purified using AMPure Beads (Beckman Coulter Genomics), quantified by Nanodrop and sent to the Plateforme d’Analyses Biomoléculaires (Institut de Biologie Intégrative et des Systèmes, Université Laval) for pyrosequencing using the 454 GS-FLX DNA Sequencer with Titanium Chemistry (Roche) and the procedure described by the manufacturer.