16S-Seq of eutrophic pond surface water
We applied a nested PCR in order to increase specificity and amplicon yield. First, the full length 16S rRNA was amplified with the primers 27F (5 M) and 1492R (5 M) on 10 ng of template using GoTaq polymerase (Promega, USA) for 30 cycles (94C30s; 50C45s; 68C90s). The PCR products were purified using ExoSAP-IT (Thermo Fisher Scientific, USA). To obtain dual index amplicons of the V4 region, a second amplification was performed on 8.5 l of PCR product using primer 515F and 806R for 30 cycles (98C10s; 55C30s; 72C45s). A final purification step using AMPure beads (Beckman Coulter, USA) was performed before samples were diluted and pooled in equimolar concentrations. The amplicons were sequenced using a MiSeq Reagent kit v2 (500-cycles) with custom primers on an Illumina Miseq platform, producing 2  250-nt paired-end reads.