16S rRNA gene sequencing
Total DNA was extracted from the stool samples using the fecal DNA isolation Kit (DP328, TIANamp Beijing, China) based on the manufacturer’s instructions, and its concentration and purity were assessed on 1% agarose gels. The 16S bacterial rRNA genes were amplified with the 515F-806R primers specific for the V4 hypervariable regions (5′-GTGCCAGCMGCCGCGGTAA-3′ and 5′-GGACTACHVGGGTWTCTA
AT-3′). All PCR reactions were performed in a 30 μL reaction mix containing 15 μl of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), PCR products were quantified and equal amounts were loaded into a 2% agarose gel for electrophoresis. The target bands were excised and analyzed by paired-end sequencing on the Illumina MiSeq platform in accordance with the manufacturer’s instruction, provided by Beijing Novogene Genomics Technology Co. Ltd. (Beijing, China).