title: amplicon seq S. virgatus microbiome: cloacal tissue
design: Two-step PCR to amplify the 16s rRNA gene V4 region. PCR1 six used pooled 515F/806R primer pairs,each with a 0-5 base pair shift and linked to a consensus sequence with a 2 bp linker. PCR 1 reaction: 7.5 uL Phusion Flash High Fidelity Master Mix, 0.15 uL forward and reverse primers, 0.75 uL BSA, 5.45 uL purified water, and 1 uL template DNA. PCR1 thermal cycler: 98C for 10 sec; 28 cycles of n 98C for 1s, 57C for 5s, and  72C for 20s;  final extension at 72C for 60s. PCR1 was performed in triplicate and pooled for PCR2. PCR2 reaction: 10 uL Phusion Flash High Fidelity Master Mix, 0.24 uL BSA, 8.01 uL of purified water, 0.75 uL of unique barcoded primer pairs (supplied by IBest Genomics Core, University of Idaho), and 1 uL of template DNA. The thermal cycling protocolmatched the protocol for PCR1, except for an annealing temperature of 51C. PCR2 was only run for 8 cycles (for a total of 36 cycles total)