title: 210_9_GG_15MAR12_Bv4v5
design: Construction protocol: The v4-v5 hypervariable regions of the SSU rRNA gene were amplified using degenerate primers. The primers were 518F (CCAGCAGCYGCGGTAAN) and 926R (CCGTCAATTCNTTTRAGT, CCGTCAATTTCTTTGAGT, and CCGTCTATTCCTTTGANT) fused to Illumina adapters and including 4 random nt followed by a 5 nt multiplexing barcode on the forward primer and 6 nt multiplexing index on the reverse primer. We generated PCR amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 10.0 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase mix (ThermoFisher), 3.0 mM MgSO4 and 0.3 uM of each primer. We added approximately 10-25 ng template DNA to each PCR and ran a no-template control for each primer pair. Amplification conditions were: initial 94C, 3 minute denaturation step; 30 cycles of 94C for 30s, 57C for 45s, and 72C for 60s; final 2 minute extension at 72C. The triplicate PCR reactions were pooled after amplification and purified using Ampure beads (1:1). Purified DNA was eluted in 30 uL of Qiagen buffer EB and size selected using Ampure (1:0.75). We assessed the quality, size and concentration of PCR products on a Perkin Elmer Caliper GX. Reads were demultiplexed by barcode and index; barcode and primer sequences are present in the paired fastq files.