title: Bacterial diversity in the water column of the Kariega estuary mouth
design: Sample Collection  Three sites were identified along the length of each of the estuaries corresponding to the upper, middle and lower reaches of the estuary.  At each site, surface water samples (200ml) were filtered through a 0.22m PES filter (Supor®-200, PALL) and sediment samples were collected in sterile 2ml eppendorf tubes.  In order to minimize degradation of genomic material, all samples were stored on dry ice immediately after collection and then later transferred to -20oC.  Physico-chemical analyses were done as described in Matcher et al. (2011).    Amplicon library preparation and multiplex pyrosequencing   Total genomic DNA was isolated from the water column and sediment from each of the sites using the PowerWater DNA and PowerSoil DNA Isolation Kit (MoBio Laboratories) respectively. Amplicon libraries were generated using the primer pair E517F (5’-CAGCAGCCGCGGTAA-3’) and E969-984 (5’-GTAAGGTTCYTCGCGT-3’) which generates amplicons including the variable regions 4 and 5 of the bacterial 16S rRNA gene. 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 538 nt PCR products were gel purified using the Zymo Gel DNA Recovery kit (Zymo Research), the respective amplicons pooled in equal amounts and subjected to emulsion PCR before sequencing using the GS Titanium Sequencing technology (454 Life Sciences, Roche Applied Sciences).