16S RNA gene sequencing
16S DNA sequencing was completed by Xiamen Treatgut Biotechnology Co. Ltd. DNA was extracted from approximately 0.25 g of fecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, CA, USA) according to the manufacturer’s instructions. The concentrations and purity of the isolated DNAs were assessed using spectrophotometry (Multiskan™ GO, Thermo Fisher Scientific, USA). The DNA extracts also were evaluated for quality by agarose (1.5%) gel electrophoresis in 1× Tris-Acetate-EDTA buffer. Samples were stored at −20˚C before being used as templates for next-generation sequencing library preparation.The V4 region of the 16S rRNA gene was amplified using the specific primers (515F [5’-GTGCCAGCMGCCGCGGTAA-3’] and 806R [5’-GGACTACNVGGGTWTCTAAT-3’]) with the barcode. The same volume of 1× loading buffer (contained SYB green) was mixed with polymerase chain reaction (PCR) products, followed by 2% agarose gel electrophoresis. The samples with the bright main strip between 400–450 bp were chosen for further experiments. PCR products were mixed in equidense ratios. Then, a mixture of PCR products was purified with GeneJET Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using TruSeq