Amplification and Sequencing of 16S rRNA

Total DNA was extracted from the 0.2 μm-pore size filter using the Meta-G-Nome^TM DNA Isolation Kit (Epicentre Biotechnologies, Madison, WI, United States) according to the manufacturer’s instructions. Extracted DNA was used as template of polymerase chain reaction (PCR) to amplify V5-V6 region (∼300 bp) of 16S rRNA gene using the forward primer FIA-787F (5′-[forward index adaptor]-ATTAGATACCCNGGTAG-3′) and reverse primer RIA-1046R (5′-[reverse index adaptor]-CGACAGCCATGCANCACCT-3′) (Cai et al., 2013 (link)). PCR was performed with two-steps to gain better reproducibility and consistent results (Berry et al., 2011 (link)). In detail, the first-step PCR conditions were an initial denaturation at 95°C for 3 min; 25 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 1 min; and a final extension at 72°C for 2 min. The second-step PCR conditions were at 95°C for 3 min, 8 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s; and a final extension at 72°C for 5 min. The amplicons from PCR were sequenced through Illumina Miseq platform, producing 2 × 300 bp paired-end reads.