Amplification and Sequencing of Microbial 16S rDNA

The metagenomic DNA was diluted and used as a template for the V3 + V4 variable region of bacterial 16S rDNA. PCR products were detected by 2% agarose gel electrophoresis and purified by the Axygen^®AxyPrep DNA gel extraction kit (Axygen Scientific Inc., Union City, CA, USA). And the quantified by QuantiFluorTM-ST Blue Fluorescence Quantification System (Promega), in which the corresponding proportion was mixed according to the sequencing volume requirement of each sample. A TransStart Fastpfu, DNA Polymerase 20-μL reaction system, was used, including 2.0 μL of 10 × buffer, 2.0 μL of 2.5 m MdNTPs, 0.8 μL of forwarding primer (5 μmol/L), 0.8 μL of reverse primer (5 μmol/L), 0.2 μL of Taq polymerase, 0.2 μL of BSA, 10 μL of template DNA, and 20 μL of ddH2O (23 (link)). The PCR products were then sequenced by the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) by Wuhan Fraser Genetic Information Co., Ltd.