Supragingival Plaque Sample
DNA was extracted from samples using MagPure Stool DNA KF Kit B (MAGEN, Guangzhou, China) /Magnetic Bead Fecal and Soil Genome Extraction Kit (MAGEN, Guangzhou, China) according to manual instruction. Transfer 100-200 mg sample to the centrifuge tube with grinding beads. Add 1 mL Buffer ATL/PVP-10, grinding the sample in the grinding machine(Shanghai Jingxin Tech, China) and incubating at 65 for 20 min. The mixture was centrifuged at 14000g for 5 minutes (Eppendorf, German). Then the supernatant was transferred to a new tube. 0.6 mL Buffer PCI add to the sample and then mix thoroughly by vortexing for 15 seconds. The mixture was centrifuged at 18213g for 10 minutes. The supernatant was transferred to deep well plate with magnetic beads binding solution (600L Buffer with magnetic beads +20L Proteinase K+5L RNase A, 700L Wash 1, 700L Wash 2, 700L Wash 3, 100L Elution Buffer. Transfer the sample to corresponding place in deep well plate of the machine (Kingfisher, Thermo Fisher, USA). Begin the corresponding program in Kingfisher. Transfer the DNA to 1.5 mL centrifuge tube for storage when the program runs out. The library was prepared by 2  Phanta Max Master Mix (VAZYME, China) polymerase, and the V3V4 variable region of 16S rDNA of bacteria was amplified by forward and reverse PCR degenerate primers 338F: ACTCCTACGGGAGGCAGCAG and 806R: GGACTACHVGGGTWTCTAATF. PCR enrichment was performed in a 50 L reaction containing 30ng template and fusion PCR primers. PCR cycling conditions were as follows: 95C for 3 minutes; 30 cycles of 95C for 15 seconds, 56C for 15 seconds, 72C for 45 seconds and final extension at 72C for 5 minutes. PCR products were purified by DNA magnetic beads (BGI, LB00V60).