Diversity and structure of benthic bacterial community from South-Eastern Pacific Ocean
The v6 hypervariable regions of the bacterial DNA of the SSU rRNA gene were amplified using forward primers 967F (CTAACCGANGAACCTYACC, CNACGCGAAGAACCTTANC, CAACGCGAAAAACCTTACC, CAACGCGCAGAACCTTACC, ATACGCGARGAACCTTACC) and reverse primers 1046R (CGACAGCCATGCANCACCT, CGACGGCCATGCANCACCT, CGACGACCATGCANCACCT, CGACAACCATGCANCACCT) fused to Roche GSFLX amplicon sequencing adapters and including 5 nt multiplexing barcodes. We generated PCR amplicons in triplicate 30 uL reaction volumes with an amplification cocktail containing 1.0 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase Mix (Pierce Nucleic Acid Technologies, Milwaukee, WI), 1.5 mM MgSO4 and 0.2 uM of each primer. We added a total of 10 ng template DNA to each PCR reaction and ran a negative, no-template control for each primer pair. Amplification conditions were performed how is described in Sogin et al., 2006, modified as follows: the initial 94C, 3 minute denaturation step was followed by 30 cycles of 94C for 30s, 57C for 60s, and 72C for 90s before a final 10 minute extension at 72C. The triplicate PCR products were pooled after amplification, purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in 12 uL of Qiagen buffer EB following the manufacturer's protocol.