Zymobiomics spike in control 2

DNA isolation, library preparation, and sequencing were carried out at CosmosID (Germantown, MD) using a vendor-optimized protocol. Briefly, plaque samples were spiked with Truepera radiovictrix, Imtechella halotolerans, and Allobacillus halotolerans using ZymoBIOMICS Spike-in Control II (Zymo Research, Irvine, CA) to enable bacterial cell number quantification. To enhance bacterial cell lysis, plaque samples were incubated with MetaPolyzyme at 35 °C for 12 h, and DNA was extracted employing the ZymoBIOMICS DNA MicroPrep with bead-beating according to the manufacturer’s instructions. The concentration of all DNA samples and libraries were determined using the Qubit dsDNA HS assay and Qubit 4 fluorometer (ThermoFisher Scientific, Waltham, MA). To construct DNA libraries, 1 ng of input genomic DNA was fragmented, amplified, and indexed utilizing Nextera XT DNA Library Preparation and Nextera Indexing Kit (Illumina, San Diego, CA). DNA libraries were purified using AMPure magnetic beads (Beckman Coulter, Brea, CA) and then normalized for equimolar pooling. Sequencing was performed with a HiSeq sequencer (Illumina), targeting a coverage of 3 − 4 million paired-end 2 × 150 bp reads.

Microbiome analysis of supragingival plaque was performed using next-generation DNA sequencing at CosmosID, Inc. (Germantown, Maryland, USA). DNA isolation, library preparation, and sequencing were carried out according to vendor-optimized protocol. Briefly, ZymoBIOMICS Spike-in Control II (Zymo Research, Irvine, CA) was added to plaque specimens to enable bacterial cell number quantification. To enhance cell lysis, plaque samples were incubated with MetaPolyzyme at 35 °C for 12 h, and DNA was extracted using ZymoBIOMICS DNA MicroPrep with bead-beating according to the manufacturer’s instructions. DNA concentrations were determined using the Qubit dsDNA HS assay and Qubit 4 fluorometer (ThermoFisher Scientific, Waltham, MA). DNA libraries were prepared using 1 ng of input genomic DNA that was fragmented, amplified, and indexed employing the Nextera XT DNA Library Preparation and Nextera Indexing Kit (Illumina, San Diego, CA). DNA libraries were purified using AMPure magnetic beads (Beckman Coulter, Brea, CA) and then normalized for equimolar pooling. Sequencing was performed using a HiSeq sequencer (Illumina), targeting a coverage of 3 − 4 million paired-end 2 × 150 bp reads.