title: Impacts of physico-chemical parameters on bacterial community structure in a gold mine impacted river: A case study of the Wonderfonteinspruit, South Africa
design: The V6 – V8 region of the 16S rRNA gene was amplified using bacterial primer pair described by Comeau et al. (2012). Three separate DNA concentrations were used for each sample: 1, 0.5 and 0.1X (concentrations ranged between 0.5 and 52 ng). Triplicate reactions for each sample were pooled and purified using Agencourt AMPure beads (Beckman Coulter, Canada, LP). The quality of pooled samples was evaluated using the Agilent DNA 7500 Chip Kit (Agilent Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies). Pyrosequencing was performed using the Roche 454 GS-FLX Titanium chemistry. Raw sequence data was quality trimmed and checked for chimeras and singletons following the MOTHUR v.1.30 pipeline.