design: hypervariable V3-V4 primers (Klindworth et al., 2013). initial stage 'amplicon PCR' targeted at amplifying the area of concern by using 12.5 ng of genomic DNA, 2x KAPA HiFi HotStart ReadyMix and 5 ?M of the above-mentioned primers. The PCR was performed at a final volume of 25 ?l by denaturing at 95 C for 3 minutes, followed by 25 cycles of 95 C at 30 seconds, 55 C at 30 seconds and 72 C at 30 seconds. A final elongation step of 72 C was included for 5 minutes. Using Agencourt AMPure XP beads amplicons were then subjected to a 'PCR clean-up' phase. After cleaning, index PCR attaching dual indexes (Nextera XT Index Kit) was performed using 5 ?l of amplicon PCR product DNA, 5 ?l of Illumina Nextera XT Index Primer 1 (N7xx), 5 ?l of Nextera XT Index Primer 2 (S5xx), 25 ?l of 2x KAPA HiFi HotStart Ready Mix, and 10 ?l of PCR-grade water The thermocycling conditions were 95C for 3 minutes followed by 8 cycles of 95C for 30 seconds, 55C for 30 seconds, 72C for 30 seconds and a final elongation step of 72C for 5 minutes. The index PCR products underwent a second step of 'PCR clean-up'. The partial 16S rRNA libraries were then quantified using a Qubit fluorometer standardized, pooled to a final concentration of 20 pM and subsequently denatured in 0.2 N NaOH solution. The pooled libraries were further diluted to a final concentration of 6 pM, spiked with 10% PhiX control and heat denatured for 2 minutes before loading samples on the reagent cartridge of MiSeq V32