*2.4. Microbial DNA Extraction*

Denture plaque and tongue surface samples suspended in PBS were collected by a centrifuge at 3000 rpm for 10 min. DNA extraction was performed by a Maxwell 16 LEV Blood DNA Kit (Promega KK, Tokyo, Japan)according to the manufacturer's instructions. DNA concentration was measured by a NanoDrop ND-2000 (Thermo Fisher Scientific KK, Tokyo, Japan). Degradation of DNA was visually checked by electrophoresis on 1% agarose gel. Degradation of DNA and contamination of RNA were checked by a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific KK, Tokyo, Japan).

Samples meeting the following criteria were used for further sequence analysis: conc > 20 ng/μL, volume ≥ 20 μL, A260/280 ≥ 1.8, and A260/230 > 1.5. In this study, all samples met the criteria.
