16S rRNA sequencing of oral microbiota

The 16S ribosomal RNA (rRNA) gene amplification procedure was divided into two polymerase chain reaction (PCR) steps. For the first PCR reaction, the V3-V4 hypervariable region of the 16S rRNA gene was amplified from the genomic DNA using primers 338F(ACTCCTACGGGAGGCAGCAG) and 806R(GGACTACHVGGGTWTCTAAT). The amplification products were purified by gel extraction (AxyPrep DNA Gel Extraction Kit, Axygen Biosciences, Union City, California, USA) according to manufacturer instructions. The concentration of the pooled libraries was determined using the Qubit quantification system. Amplicon sequencing was performed on the MiSeq PE250 platform (Illumina, San Diego, California, USA). Automated cluster generation and 2 × 250 bp paired-end sequencing with dual-index reads were performed.