16S rRNA sequences from Blackjack pond sample
A modified barcoded amplicon sequencing (bTEFAP®) procedure was used with 16S universal primers (515F: 5’-GTGCCAGCMGCCGCGGTAA-3’ and 806R: 5’-GGACTACHVGGGTWTCTAAT-3’). A single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) were used under the following conditions: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds; 53°C for 40 seconds and 72°C for 1 minute; after which a final elongation step at 72°C for 5 minutes was performed. Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). Samples were sequenced utilizing Roche 454 FLX titanium instruments and reagents and following manufacturer’s guidelines.