2.3 DNA extraction, PCR amplification, and Illumina HiSeq sequencing
Total DNA was extracted from 0.5 g of sediment using the FastDNA® Spin kit (MP Biomedicals, Santa Ana, CA) following the manufacturer’s protocol. The extracted DNA was re-suspended into 100 μL of nuclease-free water and quantified using a Qubit 4.0 fluorometer with the dsDNA Broad Range assay kit (Invitrogen, Singapore).
The V4-V5 region of the bacterial 16S rRNA gene was amplified using a universal primer pair 515F (5’-GTG YCA GCM GCC GCG GTA-3’) and 907R (5’-CCG YCA ATT YMT TTR AGT TT-3’) (Quince et al., 2011). PCR reaction was performed under the condition of 95°C for 3 min, followed by 25 cycles of 95°C for 45 s, 50°C for 60 s, 72°C for 90 s and a final cycle of 10 min at 72°C. Each sample was amplified in triplicate and pooled prior to purification with Agencourt AMpure XP beads and quantification with a Quant-iT dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). Finally, PCR products were sequenced on the Illumina NovaSeq 6000 platform at Magigene (Guangzhou, China) using the PE250 strategy.