16S rRNA Sequencing of Salivary Microbiome

We prepared libraries of the V3–V4 region of the 16S rRNA gene with KAPA HiFi HS ReadyMix (KAPA BIOSYSTEMS) using the salivary DNAs as templates according to the 16S rRNA metagenomics protocol for MiSeq system (Illumina). The PCR samples were cleaned up by AMPure XP beads (Beckman Coulter) and the read data were obtained by a MiSeq sequencer using Miseq Reagent Kit v3 and PhiX Control v3 (Illumina). The raw reads data have been registered with DNA Data Bank of Japan under the accession number DRA015154 (Table S1). The obtained read data were analyzed by the QIIME2 program (Caporaso et al., 2010 (link)) (https://qiime2.org/). After merging the pair end reads, quality check and chimera checks were conducted with the DADA2 plugin (Callahan et al., 2016 (link)) . To analyze taxonomy, a classifier was trained using SILVA 16S rRNA database (Ver. 123) (Quast et al., 2013 (link)). The samples were rarefied at 10,000 depths of sequences.