Extraction and Sequencing of Microbial DNA from Fecal Samples

Microbial DNAs from the fecal samples were extracted from approximately 200 mg of each stool sample using the QIAamp PowerFecal Pro DNA kit (Qiagen, Valencia, CA, USA) by following the manufacturer’s protocol. To construct sequencing libraries, the V4 to the V5 hypervariable region in the 16S rRNA gene was amplified using the 515 F (5′-barcode-GTGCCAGCMGCCGCGGTAA-3′) and 907 R (5′-barcode-CCGYCAATTCMTTTRAGTTT-3′) indexed reverse primers. The specific PCR conditions are described in a previous study [32 (link)]. Afterward, gel electrophoresis and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to confirm the quality and quantity of the library DNA. The library DNAs were then pooled equally and then purified using an AMPure XP bead (Beckman Coulter, Brea, CA, USA). The libraries were sequenced using an Ion Torrent PGM platform for 1250 flows with the Ion PGM^TM Hi Q Sequencing Kit (Thermo Fisher Scientific, USA).