title: 18S rRNA gene of pond metagenome
design: Sequencing of PCR amplicons of 18S rRNA was conducted with the Illumina MiSeq platform (Illumina, San Diego, CA, USA) targeting the V9 hyper variable region (forward primer, 1391F, 5'-GTACACACCGCCCGTC-3'; reverse primer, EukBR, 5'-TGATCCTTCTGCAGGTTCACCTAC-3') for 2250-bp paired-end sequencing. PCR amplification was performed using Roche FastStart High Fidelity PCR System in 50ul reactions under the following conditions: initial denaturation at 95C for 2min, followed by 36 cycles of 95C for 30s, 55C for 30s, 72C for 1 min, and a final extension step of 5min at 72C. The PCR products were purified and normalised to 20ng each using the SequalPrep Normalization Kit . The PCR amplicons from all samples were pooled in equimolar ratios. The run was performed using a 4pM sample library spiked with 4pM PhiX to a final concentration of 12% following the method of Schloss and Kozich.