Experimental model and subject details

All the adult Chinese individuals in this 4D-SZ cohort were recruited for a multi-omics study as previously reported.^15,16,17,18 This study at present included a total of 4,478 individuals, of which 3,504 had saliva samples and 3,694 had tongue dorsum samples (3,165 individuals having both sample types) for whole metagenomic sequencing (Table S1). The cohort could be divided into two independent parts, namely discovery and replication. In the discovery cohort, 2,984 individuals were enlisted with blood samples in the city of Shenzhen for high-depth whole genome sequencing (a mean depth of 33X), of which 2,017 had tongue dorsum and 1,915 had salivary samples for whole metagenomic sequencing. In the replication cohort, blood samples were collected from 1,494 individuals with low-depth whole genome sequencing (a mean depth of 9X), out of which 1,333 had tongue dorsum samples and 1,299 had salivary samples for metagenomic sequencing (Table S1). The replication cohort was designed in the same manner but organized at smaller scales in multiple cities (Wuhan, Qingdao, etc.) in China. The protocols for blood and oral collection, as well as the whole genome and metagenomic sequencing, were similar to our previous literature.^15,16,17,18 For the blood sample, DNA was extracted using MagPure Buffy Coat DNA Midi KF Kit (no. D3537-02) according to the manufacturer’s protocol. Tongue dorsum and salivary samples were collected with the MGIEasy kit. For the salivary sample, a 2x concentration of stabilizing reagent kit was used and 2 mL saliva was collected. DNA of oral samples was extracted using MagPure Stool DNA KF Kit B (no. MD5115-02B). The DNA concentrations from blood and oral samples were estimated by Qubit (Invitrogen). 500 ng of input DNA from blood and oral samples were used for library preparation and then processed for paired-end 100 bp sequencing using the BGISEQ-500 platform.⁸²

The study was approved by the Institutional Review Boards (IRB) at BGI-Shenzhen, and all participants provided written informed consent at enrollment.