Comprehensive Microbiome Profiling

Microbiome samples were obtained from participants’ tongue plaques and stool samples for oral and gut microbiomes, respectively, and were stored at − 80 °C until use. The detailed library preparation method, including PCR conditions, has been described in a previous paper^{27 (link)}. Briefly, the samples were mixed with zirconia beads and lysed using a FastPrep FP100A instrument (MP Biomedicals, Santa Ana, CA, USA). DNA was extracted from the bead-treated suspensions using a Magtration System 12GC and GC series MagDEA DNA 200 (Precision System Science, Chiba, Japan) and amplified. The 16S rRNA gene amplicons covering the V3–V4 region were sequenced using Pro341F and Pro805R primers. Sequencing was performed using a paired-end, 2 × 250-base pair cycle run on an Illumina MiSeq sequencing system. Shotgun metagenomic sequencing was performed on the HiSeq2500 instrument (Illumina, San Diego, CA, USA) with 101 bp paired-end reads.