title: 16s Bv6 amplicon sequencing of sample TWNCO2_2547m
design: The v6 hypervariable region of the bacterial SSU rRNA gene was amplified using degenerate primers. The primers were 967F (CTAACCGANGAACCTYACC CNACGCGAAGAACCTTANC CAACGCGMARAACCTTACC ATACGCGARGAACCTTACC) and 1064R (CGACRRCCATGCANCACCT). Amplification was done in two rounds; the first with the 16S-only primers and the second with the same sequences fused to Illumina adapters. The forward primers included a 5 nt multiplexing barcode and the reverse a 6 nt index. We generated PCR amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 1.3 U Platinum Taq Hi-Fidelity Polymerase (Invitrogen, Carlsbad, CA), 1X Hi-Fidelity buffer, 200 uM dNTP PurePeak DNA polymerase mix (ThermoFisher), 2 mM MgSO4 and 0.2 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; 25 cycles of 94C for 30s, 60C for 45s, and 72C for 60s; final 2 minute extension at 72C. The triplicate PCR reactions were pooled after amplification and purified using Qiagen MinElute plates. The second round of PCR used 4 ul of the step 1 eluate as template for 5 cycles followed by clean up, PicoGreen quantitation and Sage PippinPrep size selection.