Environmental master mix version 2.0 pcr buffer

Both forward (J1Fi5) and reverse (J1Ri7 or J2Ri7) barcoded primers (Table 1) were introduced during a second round PCR step. The barcoded primer (J1Fi5) included a sequencing adapter at the 5′ end and JS15 sequence at the 3′ end (Table 1). The barcoded primer (J1Ri7 or J2Ri7) included a sequencing adapter, a unique single index 8 base barcode at the 5′ end and JS15 sequence at the 3′ end (Table 1). Single-indexed PCR primers (i7) with unique indexes (that may be useful for multiplexing samples) were used. An 8-base single-indexed sequencing run workflow was adopted using indexes aaccgcgg or ggttataa (Table 1). These barcoded PCR primers were designed to be less than 60 bases. PCR reactions were performed in a 50 μL reaction volume with 5 μL of amplified product from the PCR enrichment step, 1.25 μL of i5 primer at 10 μM, 1.25 μL of i7 primer at 10 μM, 25 μL of Environmental Master Mix (version 2.0) PCR buffer (Life Technologies, Grand Island, NY) and 17.5 μL of nuclease-free water. A short PCR amplification reaction was carried out with an initial denaturation step for 10 min at 95 °C followed by 25 cycles of 30 s at 95 °C, 30 s at 60 °C, 30 s at 68 °C. A final extension step at 68 °C for 5 min was included. The amplification products were purified with SPRIselect beads at a 1:0.8 ratio. After cleanup, the final barcoded DNA amplicons ranged between 300-700 bp.