Microbial diversity along the south west coast of India influenced by co-existing upwelling and mud banks
PCR products were molar-normalized, pooled into a single tube, and purified to a minimum of 300 bp using PureLink PCR Purification kit (Invitrogen, CA, USA). The purified, pooled sample was then diluted to 4 nM final concentration using Resuspension Buffer (RSB – Illumina, CA, USA). The sample was denatured for 5 minutes and neutralized using 0.2 N NaOH and HT1 Buffer, respectively (Illumina, CA, USA). It was then pooled with other libraries prepared for NGS in a ratio dependent on amplicon size/total panel size, desired sequencing depth, and number of samples pooled in each sub-library. Pooled libraries were further diluted down to a final 15 pM library and spiked with 5% phiX (Illumina, CA, USA) as a control and diversity enhancer. Samples were then loaded into an Illumina MiSeq v3 cartridge (Illumina, CA, USA) and run in 2*300 mode on an Illumina MiSeq next generation sequencer (Illumina, CA, USA) (Ramanan et al. 2016).