16S rRNA gene sequencing of brine mesocosm T5months Control2
Amplicons of the V4 hypervariable region of the 16S rRNA gene were generated using the Roche FastStart High Fidelity PCR System (Roche Diagnostics Ltd, Burgess Hill, UK). DNA was initially denatured at 95C for 2min, followed by 37 cycles of denaturation, annealing, and extension (30 s at 95C, 30 s at 55C, 1 min at 72C), followed by a final extension of 72C for 3 min. PCR amplicons were purified and normalized to 20 ng using the SequalPrep Normalization Kit (Fisher Scientific, Loughborough, UK) before pooling in equimolar ratios. Sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, USA) using paired-end chemistry and the 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) primers and a 6 pM library spiked with 6 pM PhiX to a final concentration of 20 % (Kozich et al., 2013).