16S rRNA sequencing
Barcoded 16S rRNA amplicon (V3–V4 regions) sequencing was performed using Illumina MiSeq technology (Personalbio, Shanghai, China) and primers F (5′-ACTCCTACGGGAGGCAGCA-3′) and R (5′-TCGGACTACHVGGGTWTCTAAT-3′). A unique 12-mer tag for each DNA sample was added to the 5′-end of both primers to allow pooling of multiple samples in one run. The PCR products were visualized on a 3% agarose gel, gel purified, quantified with a Pico-Green kit, pooled in an equimolar ratio, assessed using an Agilent BioAnalyzer 2100 (Invitrogen, Carlsbad, CA), and sequenced. Sequences were trimmed using Trimmomatic 2 based on a quality score of 20, and pair-end reads were merged into longer reads using FLASH 3. Unqualified sequences were removed if they were too short or if they contained ambiguous residues. Operational taxonomic units (OTUs) were clustered using Usearch (version 7.1, Before bioinformatic analysis, sequencing reads of all samples were standardized by rarefying OTU tables to the minimum read number. Analyses were performed using the online Majorbio Cloud Platform (