Metagenomic Sequencing of human gut microbiota
Fecal DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturers instructions. Approximately 200 mg of fecal samples were homogenized in extraction buffer with a TissueLyser LT (Qiagen, Germany), heated at 95 C for 10 minutes, centrifuged, and treated with Proteinase K. DNA was eluted in 100 L Buffer ATE. Extracted DNA was quantified using Qubit (Invitrogen, USA). The 16S rRNA gene amplicon library preparation and sequencing were performed by Microbiome Insights (Vancouver, Canada). For library preparation, Bacterial 16S rRNA genes were PCR-amplified with dual-barcoded primers targeting the V4 region (515F 5-GTGCCAGCMGCCGCGGTAA-3, and 806R 5-GGACTACHVGGGTWTCTAAT-3), as per the protocol of Kozich et al. [17]. Amplicons were sequenced with an Illumina MiSeq using the 300-bp paired-end kit (v.3).