Swartkops Estuary - bacterial diversity in the water column and sediment
Primer pair E517F (5’-CAGCAGCCGCGGTAA-3’) and E969-984 (5’-GTAAGGTTCYTCGCGT-3’) were used to amplify variable regions 4 and 5 of the bacterial 16S rRNA gene and barcoding of the amplicons from each sample site was done using Multiplex Identifier Tags.  PCR amplification of the 16S rRNA gene fragments from each sample was carried out as described previously (Matcher et al., 2011). The resultant PCR products (approximately 540 bp in size) were gel-purified (Zymo Gel DNA Recovery kit, Zymo Research) and the respective amplicons pooled in equal amounts.  The amplicon library was then subjected to emulsion PCR before pyrosequencing using the GS Titanium Sequencer according to the manufacturer’s specifications (454 Life Sciences, Roche)