16S rDNA V3-V4 amplicon sequencing of oral sample from healthy control
The V3-V4 hypervariable region of the 16S rDNA gene was amplified using forward primer 5-CCTACGGGNBGCASCAG-3 and reverse primer 5-GGACTACNVGGGTWTCTAAT-3. Unstimulated saliva was collected in the morning before breakfast. DNA was extracted using QIAamp DNA Fecal Mini Kit. PCR used 15 uL Phusion High-Fidelity PCR Master Mix, 0.2 uM forward and reverse primers, and approximately 10 ng template DNA. Thermal cycling: 98C 1 min; 30 cycles of 98C 10s, 50C 30s, 72C 30s; final extension 72C 5 min. Paired-end 250 bp sequencing was performed on an Illumina HiSeq 2500 platform. Raw reads were assembled using FLASH, and primers and low-quality reads were removed with Cutadapt.