Evaluation of Impact of Coastal pollution on Human health: A prospective cohort study
High quality genomic DNA (gDNA) was isolated using  QIAamp PowerFecal Pro DNA Kit (Qiagen). gDNA samples were subjected to a quality check using a spectrophotometer and the A260/280 ratio were recorded followed by a 16S rRNA gene general PCR to check if gene amplification was possible for each sample received. Samples that failed in the PCR were excluded irrespective of them qualifying spectrophotometric QC. This was done to assure success of library prep. The PCR successful samples were quantified with Qubit fluorometer using a Qubit broad range dsDNA quantification kit (Thermo Fisher Scientific). The samples were then diluted to 5ng/L in Low TE and subjected to library preparation workflow for the 16S rRNA gene V3-V4 region using the Illumina demonstrated protocol for 16S Metagenomic sequencing. The protocol uses a phased primer strategy as outlined by Fadrosh et al. (2014). Indexed libraries were purified and pooled at equimolar concentrations of 4nM. The pool was further validated using an Agilent Tapestation 4200 high sensitivity D1000 screentape (Agilent) and quantified using the Qubit fluorometer high sensitivity ds DNA kit (Thermo Fisher Scientific). The pooled library was sequenced on an Illumina MiSeq instrument using the 2x300 base chemistry of the v3 reagent kit as per manufacturers instructions. The reads produced were demultiplexed on board the sequencer and raw fastq files were generated.