Main_June_17_fun
NGS sequencing of microorganisms present in a high pH spent nuclear fuel pond
Sequencing of PCR  amplicons of the ITS2 region was conducted with the Illumina MiSeq platform  (Illumina, San Diego, CA, USA) targeting the ITS region of the 5.8S and large  subunit (LSU) ribosomal genes (forward primer, ITS4F,  5'-AGCCTCCGCTTATTGATATGCTTAART-3'; reverse primer, 5.8SR,  5'-AACTTTYRRCAAYGGATCWCT-3') for 2300-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, 56C for 45s, 72C for 2 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.
Main_June_17_fun
SUB5263014
Main_June_17_fun