Microbiome of produced water from Brazilian oilfields
The V3 and V4 variable region of the 16S rRNA was amplified by PCR, using the primers 338F (Whiteley & Bailey, 2000) and 806R (Caporaso et al., 2011) with barcode on the forward primer, in a 30 cycle PCR (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 minutes was performed. After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples are pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. Paired-end Ssequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturers guidelines. After sequencing, the two reads from the paired end sequencing were joined together after q25 trimming of the ends, with MrDNA pipeline.