Fecal DNA Extraction and Characterization

Fecal samples were collected on the same day of blood collection as in our previous studies [11 (link),22 (link)]. In brief, the fecal samples were collected and stored at −80 °C for up to three days before processing. A stool DNA Extraction kit (Topgen Biotechnology Co., Ltd., Kaohsiung, Taiwan) was used to extract bacterial DNA. The fecal samples weighted to 50 to 100 mg were supplemented using a preceding bead beating (45 s; 3450 oscillations/min). The subsequent steps of DNA extraction were performed according to the manufacturer’s protocol. DNA concentration and quality were assessed using the Colibri Microvolume spectrophotometer (Titertek Berthold, Pforzheim, Germany). Extracted DNA samples were immediately stored at −20 °C before use.