Fecal DNA Extraction and High-Throughput Sequencing

Fresh fecal pellets (0.3 g) of each participant were used for microbial DNA extraction. Fecal microbial DNA was extracted using a PowerSoil DNA isolation kit (Mobio, United States). DNA concentration and quality were checked using a NanoDrop spectrophotometer (Thermo Fisher Scientific, United States).

The V4–V5 hypervariable region of the prokaryotic 16S rRNA gene was amplified using the universal primer pair 515F (5′-GTGYCAGCMGCCGCGGTA-3′) and 909R (5′-CCCCGYCAATTCMTTTRAGT-3′), with a 12-nt sample-specific barcode sequence included at the 5′-end of the 515F sequence to distinguish samples (Ni et al., 2017; Huang et al., 2018; Xiang et al., 2018). Polymerase chain reaction (PCR) was performed, and amplicons were sequenced using a MiSeq system at Guangdong Meilikang Bio-Science, Ltd. (China), as described previously (Huang et al., 2018; Xiang et al., 2018).

The raw sequences were merged using FLASH-1.2.8 software (Magoc and Salzberg, 2011) and processed using the QIIME Pipeline 1.9.0 with default parameters (Caporaso et al., 2010). Chimeric sequences were identified and removed using the Uchime algorithm before further analysis (Edgar et al., 2011). The high-quality sequences were clustered into OTUs at 97% identity using UPARSE (Edgar, 2013). Taxonomic assignments of each OTU were determined using the RDP classifier (Wang et al., 2007).