GSM8880728: C11_saliva_Healthy; human oral metagenome; OTHER
DNA extraction from oral specimens Oral bacteria were collected from the periodontal pockets and tongue tissue during oral care. In the edentulous jaw, we collected bacteria only from the tongue, whereas in the case of the teeth, we collected bacteria from the periodontal pockets. The QIAamp DNA Mini Kit (Qiagen) was used to extract genomic DNA from the oral samples. Fecal samples were collected by the participants themselves following the manufacturer's instructions. DNA was extracted from 200 mg of stool using a QIAamp DNA Stool Mini Kit (Qiagen). After determining the concentration of genomic DNA extracted from the samples using Nanodrop one (Thermo Fisher Scientific), 6.25 ng DNA was added to 25 μl of 2x KAPA HS HiFi ready mix (KAPA Biosystem). The DNA mixture was amplified using a PCR thermal cycler (C1000 Touch Thermal Cycler, Bio-Rad) programmed with an initial denaturation of 3 minutes at 95°C, followed by 25 cycles of denaturation at 95°C for 30 seconds, annealing at the temperature of 72°C for 30 seconds, and extension at 72°C for 30 seconds with the following primer pair for the V3-V4 region of 16S rRNA. Forward primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 16S amplicon Reverse primer: TCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAAT CCA. The barcode and adaptor sequence were inserted into the products by the Nextera XT Index Kit (Illumina) using a C1000 Touch Thermal Cycler (Bio-rad) programmed with an initial denaturation of 3 min at 95°C, followed by 8 cycles of denaturation at 95°C for 30 sec, annealing at the temperature of 55°C for 30 sec, and extension at 72°C for 5 min. Product concentrations were confirmed using a Qubit (Thermo Fisher Scientific). The libraries were pooled to equal molecular amounts and analyzed on an Illumina Mi-Seq DNA sequencer with a 300 bp paired-end cycle sequencing kit (Illumina).