2.2. Faecal microbiota analysis

Sample collection: Each participant provided a fresh stool sample that was delivered immediately to the laboratory in an ice bag. It was then divided into five aliquots of 200 mg and immediately stored at −80°C in the laboratory.

DNA extraction: Extraction of DNA from stool was performed following the protocol of the QIAamp DNA stool minikit (Qiagen).

PCR amplification and sequencing: PCR amplification and sequencing were conducted at Realbio Genomics Institute (Shanghai, China). The V3‐V4 region of the bacteria 16S ribosomal RNA genes was amplified by PCR using primers 341F 5′‐CCTACGGGRSGCAGCAG)‐3′ and 806R 5′‐GGACTACVVGGGTATCTAATC‐3′. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) according to the manufacturer's instructions and quantified using Qubit®2.0 (Invitrogen). After preparation of library, these tags were sequenced on MiSeq platform (Illumina, Inc) for paired end reads of 250 bp, which were overlapped on their three ends for concatenation into original longer tags.

Process of sequencing data: Tags, trimmed of barcodes and primers, were further checked on their rest lengths and average base quality. 16S tags were restricted between 220 and 500 bp such that the average Phred score of bases was no worse than 20 (Q20) and no more than three ambiguous N. Operational taxonomic units (OTUs) were clustered with 97% similarity using UPARSE (http://drive5.com/uparse/), and chimeric sequences were identified and removed using Userach (version 7.0). Each representative tags was assigned to a taxa by RDP Classifier (http://rdp.cme.msu.edu/) against the RDP database (http://rdp.cme.msu.edu/) using confidence threshold of 0.8. OTU profiling table, and alpha/beta diversity analyses were also achieved by python scripts of Qiime.