title: 16s Amplicon sequencing of Pocillopora verrucosa
design: The V3 and V4 regions of the 16s rRNA gene were amplified from the extracted DNA by Polymerase Chain Reaction (PCR) using the 341F and 785R primer pair with Illumina MiSeq adapter overhang sequences added to the gene-specific nucleotide sequences. The sequences of the primers were: 341F = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 785R = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. The reaction was prepared with the KAPA HiFi HotStart ReadyMix (Roche, Switzerland), with 5 M of each primer, 12.5 ng of DNA and 12.5 L of the Mix, diluted with DNAse-free water to a volume of 25 L. The temperature profile for amplification was as follows:  one cycle of five minutes at 92 oC, followed by 30 cycles of 30 seconds at 92 oC, 30 seconds at 62 oC and 30 seconds at 72 oC, followed by one cycle at 72 oC. For each sample, reactions were done in triplicate and then combined in a single pool. All sequences were checked by agarose gel electrophoresis for correct amplification before pooling. The resulting PCR product was sent to Macrogen (Seoul, South Korea) for DNA sequencing using the MiSeq sequencing platform.