design: The extracted DNA was used as template for PCR amplification of bacterial 16S rRNA (V3-V4) genes with the barcoded primers and Takara Ex Taq (Takara). The Amplicon quality was visualized using agarose gel electrophoresis. The PCR products purified with AMPure XP beads (Agencourt) and amplified for another round of PCR. After purified with the AMPure XP beads again, the final amplicon was quantified using Qubit dsDNA Assay Kit (Thermo Fisher Scientific, USA). The concentrations were then adjusted for sequencing.