title: Diamante Lake Red Biofilms Bacterial Diversity
design: The V4 hypervariable region of the 16S rDNA gene was amplified using F563-R802 universal primers that are reliable amplifying Bacteria but not Archaea. Primer sequences from 5’ to 3’ were the following: 563F: AYTGGGYDTAAAGNG and 802R: Equimolar pool of TACCRGGGTHTCTAATCC, TACCAGAGTATCTAATTC, CTACDSRGGTMTCTAATC, TACNVGGGTATCTAATCC and were obtained from RDP’s Pyrosequencing Pipeline: http://pyro.cme.msu.edu/pyro/help. The sample was amplified in two rounds of PCR. First round was performed with 563F-802R 16S rRNA primers fused at 5´end to a TAG sequence (TAG-fw: CACGACGTTGTAAAACGAC in primer forward and TAG-Rv: CAGGAAACAGCTATGACC in primer reverse). Three independent PCRs were performed and then pooled to reduce PCR bias. The PCR mix (final volume 25 ul) contained 2.5 ul FastStart High Fidelity 10X Reaction Buffer (Roche Applied Science, Mannheim, Germany), 20 ng of the same DNA template used for Metagenomic shotgun library, 0.4 uM of each primer, 1.25 U FastStart High Fidelity Enzyme Blend (Roche Applied Science), and 0.2 mM DNTPs. The PCR conditions were 95 °C for 5 minutes for initial denaturalization, followed by 95 °C for 45 seconds, 57 °C for 45 seconds, 72 °C for 60 seconds in 30 cycles, and a final elongation step at 72°C for 4 minutes. Two negative control reactions containing all components without PCR template were performed. A second round of PCR was performed to add Roche 454 adaptors and a specific sequence identifier (MID) as DLRB sample was sequenced together with samples from other projects. Forward primers used in second round were (from 5´to 3´): Roche 454-A adaptor, MID, TAG-fw and Reverse primer was: Roche 454-B adaptor, TAG-rv. Two independent PCRs per sample were performed using first PCRs pool as template and products were finally pooled and purified using AMPure Beads (Beckman Coulter, Cat: A63880) to generate the final amplicon library for 454-FLX sequencer.