BE326FW250111BH2 bacterial microbiome v6 survey
"Construction protocol: 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 5 nt multiplexing barcodes and the reverse a 6 nt index. We generated PCR         amplicons in triplicate 33 uL reaction volumes with an amplification cocktail containing 4.0         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."