Sterile conical polypropylene tube with flat top screw cap

A saliva sample collection procedure was conducted according to the microbiome sampling protocol of the Human Microbiome Project (McInnes and Cutting, 2010 ). All saliva samples were collected following the same protocol. Saliva samples of the OSA group were collected at 6:00–7:00 a.m. right after finishing PSG monitoring at the Sleep Center of Beijing Children’s Hospital. Participants were asked to sit in a comfortable position and not to brush their teeth, drink, or eat to minimize the potential risk of contamination. The participants were then instructed to let saliva collect in the mouth for 1 min and then drool into the labeled 50-ml collection tube (Falcon, sterile conical polypropylene tube with flat-top screw cap). This process may be repeated multiple times in order to collect 1.5 ml of unstimulated saliva. Saliva samples of the control group were collected following the same protocol as mentioned above at the participants’ own homes. After collection, the samples were immediately placed on ice and transferred to the laboratory within 1 h. All the samples were equalized to the volumes of 1 ml by pipettes and then were centrifuged at 10,000 ×g for 10 min at 4°C. The pellets were stored at −80°C before DNA extraction.

Fecal samples were collected using the Fisherbrand™ Commode Specimen Collection System (Thermo Fisher Scientific, Waltham, MA, United States). Saliva samples were collected by asking each subject to let saliva collect in their mouth for at least 1 min. The subject was then asked to drool into a labeled 50 ml collection tube (Falcon, sterile conical polypropylene tube with flat-top screw cap). This process was repeated multiple times to collect larger volumes of saliva (2–5 ml). Oral cavity microbiome samples were from the dorsum of the tongue using Catch-All™ Sample Collection Swabs and swabbing 1 cm² of the center of the tongue for 5 s. Immediately after swabbing, each swab was swirled in MO BIO’s PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, United States) collection tube with 750 ul prefilled collection buffer (Tube C1). The swab sponge was pressed against the tube wall multiple times for 20 s to ensure the transfer of bacteria from the swab to the solution. The specimen in the collection tube was kept cold until ready for processing.