freshwater metagenome: Sample KA_water_09_18_13
"Fusion primers contain Truseq adapter sequences (bridge adapter, sequencing primer binding sites and include Forward Primer (518F) CCAGCAGCYGCGGTAAN and Reverse Primers (926R) CCGTCAATTCNTTTRAGT, CCGTCAATTTCTTTGAGT, CCGTCTATTCCTTTGAT. F/R primer pools are mixed at a ratio of 8:1:1 uM in respective order.The PCR reaction contains: 1X HiFi buffer, 3 mM MgSO4, 0.2 mM dNTPs, 0.3 uM combined primers, 10 units Platinum HiFi, 5-20 ng DNA template, 100 ul total volume. The reaction is run for 3 min at 94*; 30 cycles of 30 sec 94*, 45 sec 57*, 1 min 72*, and 2 min at 72*.The reaction volume is divided into three replicate 33 ul reactions. We include a no-template negative control for each MID. The three replicates are pooled and we check 1 ul of the pool and the negative control on a LabChip GX. We clean the reactions and removed unwanted small products using Ampure beads. The final multiplex pool is size selected with 0.75X volumes of Ampure and quantitated using KapaBiosystems qPCR before clustering on the flowcell."