16S rRNA gene sequencing of biofilm grown from Athabasca river sediments
Partial 16S rRNA gene amplicons were produced using the universal primers E786 (5’- GATTAGATACCCTGGTAG-3’) and U926 (5’-CCGTCAATTCCTTTRAGTTT-3’) (1) containing the 10-bp multiplex identifiers (MID) and adaptor sequences for Ion Torrent sequencing described previously (3, 54). Reactions were performed in 25-μl volumes containing 1 μl of template DNA, 0.3 μM each primer, 0.4 mg/ml of BSA, 0.2mM of dNTPs and 0.05 U/μl of rTaq DNA polymerase (GE Healthcare, Baie d’Urfé, Canada). Cycling conditions involved an initial 5 min denaturing step at 95°C, followed by 25 cycles of 30 s at 95°C, 30 s at 55°C, and 45 s at 72°C, and a final elongation step of 3 min at 72°C. All PCR products were purified on agarose gels using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and quantified using the PicoGreen dsDNA quantitation assay (Invitrogen, Carlsbad, CA). All the 36 amplification products from the different samples were pooled in an equimolar ratio and sequenced together. A total of 3.50x107 molecules were used in an emulsion PCR using the Ion OneTouchTM 200 Template Kit (Life Technologies) and the OneTouchTM instruments (Life Technologies) according to the manufacturer’s protocol. The sequencing of the pooled library was done using the Personal Genome MachineTM (PGM) system and a 314 chip with the Ion Sequencing 200 kit according to manufacturer’s protocol.