2.2. Microbiome Analysis

DNA extraction from a stool sample was performed using a QIAamp DNA stool mini kit (Qiagen, Valencia, CA, USA) and bacterial 16S rRNA genes were amplified using an Ion 16S™ Metagenomics Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The kit included 2 primer tubes with 3 primer sets that amplify the hypervariable regions of 16S rRNA (V2, V4, V8 and V3, V6–7, V9, respectively). After PCR amplicons were purified using Agencourt AMPure^® XP beads (Beckman Coulter, Indianapolis, IN, USA), sequencing libraries were prepared using an Ion Plus Fragment Library Kit and Ion Xpress™ Barcode Adapters (ThermoFisher Scientific, Waltham, MA, USA). Prepared libraries were quantified using a High Sensitivity DNA kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Template preparation and sequencing were performed using the Ion Chef™ System and Ion S5™ XL system with Ion 530™ Chip Kit (ThermoFisher Scientific, Waltham, MA, USA). After filtering out low quality and polyclonal reads, and trimming any adaptor sequences at the 3′ end, the sequencing data were exported as FASTQ files and were processed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline 1.9.1 [19]. After quality filtering, 26,634,236 sequences were obtained, with a mean of 89,165 sequences per sample (min: 16,585, max: 335,275). Operational taxonomic units (OTUs) were clustered based on 97% sequence similarity with at least 10 identical sequences and assigned against the curated Greengenes v.13.8 reference database at the QIIME web site (http://qiime.org/home_static/dataFiles.html).

Alpha and beta diversity measures were calculated by QIIME [20]. Alpha diversity assessment was based on observed OTUs, including unidentified OTUs, and Chao1 index. Microbial diversity was visualized using principal coordinate analysis (PCoA) and microbial diversity between samples was assessed by qualitative (unweighted UniFrac) and quantitative (weighted UniFrac) distances. The mean relative abundance (percentage among all reads) in each group was compared at the phylum and genus levels.