16s rRNA amplicon sequencing
16s rRNA amplicon sequencing was performed using saliva collected from 29 participants. DNA was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA was quantified using a Quant-IT PicoGreen (Invitrogen, Waltham, MA, USA). Sequencing libraries were prepared according to Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 regions. The input 2 ng gDNA was amplified by polymerase chain reaction (PCR) with 5× reaction buffer, 1 mM dNTP mix, 500 nM of each of the universal F/R PCR primers, and Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA). The universal primer pair with Illumina adapter overhang sequences used for the first amplification was as follows:V3-F: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′.V4-R: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′.The first PCR product was purified using AMPure beads (Agencourt Bioscience, Beverly, MA, USA). Following purification, 2 μL of the PCR product was amplified for final library construction containing the index using the NexteraXT Indexed Primer (Illumina, San Diego, CA, USA). The PCR products were purified using AMPure beads. The final purified product was quantified according to the quantitative real-time (q)-PCR Quantification Protocol Guide (Illumina sequencing platforms) using the TapeStation D1000 ScreenTape (Agilent Technologies). Paired-end (2 × 300 bp) sequencing was performed using the MiSeq platform (Illumina).Sequenced datasets were classified by index sequencing and produced in a paired-end FASTQ format. Adapter and primer sequences were removed using Cutadapt (version 3.2) (