16S rRNA sequencing of site filtered water
DNA extracted from samples were sequenced using 16S rRNA tagged amplicon illumina sequencing by MR-DNA (Molecular Research LP, Shallowater, TX). Using the 16S rRNA V4 variable region PCR primers 515/806 a 28 cycle PCR was performed with the conditions: 94C for 3 minutes hotstart, followed by 28 cycles of 94C for 30 seconds denaturation step, annealing step at 53C for 40 seconds and elongation step at 72C for 1 minute, with a final elongation step at 72C for 5 minutes. Samples were pooled in equal proportions and purified using Ampure XP beads (Agencourt Bioscience Corporation, Beverly, MA). Sequencing was performed using Miseq following manufacturers guidelines.
SUB6419504
04.DeMMO4.steri.083117