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).