title: NGS sequencing of microorganisms present in a high pH spent nuclear fuel pond
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.
library_name: Main_Sept_17_euk