title: Probiotic formulation for oral health
design: DNA was extracted from the bacterial culture using the Alexgen Bacterial DNA Extraction Kit, and its quantity was determined using a fluorometer (Qubit4.0). The DNA sample was amplified using a V3-V4 primer set and verified using gel electrophoresis. The PCR product was then re-amplified using V3F and V4R primers with an overhang adapter. The primers used were as follows: V3F, TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG; V4R, GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. The PCR products were purified using Ampure XP beads and the purified amplicons were indexed using the Nextera XT index kit. After another round of purification with AmPure XP beads, the indexed amplicons were examined using an Agilent Tape Station 4150. The libraries were quantified using the Qubit HS and qPCR mix and then pooled for sequencing. Pooled PCR products were sequenced by hybridizing them onto the oligonucleotide-coated surface of a flow cell. DNA template copies were immobilized and amplified to generate clonal DNA clusters. Sequencing was performed using a PE 300 kit for 16S rRNA sequencing on the ILLUMINA MiSeq platform.