title: Human microbiome 16s RNA sequencing_Stool_control
design: To obtain oral microbiome samples, participants were asked to produce 1-3 mL of saliva after refraining from eating, drinking and oral hygiene practice for 1 hour. Samples were collected with sterile DNA- and RNA-free 15 ml Falcon tubes and immediately frozen at -80C. De-identified and coded samples were shipped to Precidiag Inc. for DNA extraction and sequencing on dry ice. Stool samples were collected by the participants at home under the supervision of trained parents with HR-Easy Stool Collection Kit (Precidiag, Inc.) and stored at room temperature, followed by de-identification and shipment to Precidiag CLIA-certified laboratory for DNA extraction and sequencing analysis. HR-Easy Stool Collection Kit provides a superior method for collection, storage and stabilizing stool samples for microbiome study at ambient temperature for up to a month with minimal alterations when compared with freshly-collected samples (Yu et al., manuscript in preparation). Microbial DNA was then extracted using HR-Easy Fecal DNA Kit (Precidiag, Inc.) according to manufacturers instructions and DNA samples were carefully quantified with Nanodrop Spectrophotometer. A260/A280 ratios were also measured to confirm high-purity DNA yield. DNA samples were frozen at -20C until use.