title: 16S rRNA V1V3
design: Subgingival plaque samples were sent to the Forsyth Oral Microbiome Core for DNA extraction. A modified DNA isolation protocol was used to lyse plaque samples with overnight incubation with Ready-Lyse Lysozyme Solution (Lucigen Corporation, Cat. No. R1802M) followed by using a MasterPure DNA Purification Kit (Lucigen Corporation, Cat. No. MCD85201). DNA samples were sent to Zymo Research (Irvine, CA) for sequencing (V1V3 region). The 16S rRNA gene-targeted sequencing was performed using the Quick-16S NGS library preparation kit (Zymo Research, Irvine, CA). The 16S primers were used to amplify V1-V3 region of the 16S rRNA gene. The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore prevent PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with Select-a-Size DNA Clean & Concentrator (Zymo Research, Irvine, CA), then quantified with TapeStation and Qubit. The final library was sequenced on Illumina MiSeq with a v3 reagent kit (600 cycles). The sequencing was performed with > 10% PhiX spike-in. The 16S sequences were processed further using a custom in-house pipeline that takes advantage of both the advanced amplicon de-noising algorithm of the recently available DADA2 program and the extensive collection of 16S sequences in the Human Oral Microbiome Database (HOMD) housed at Forsyth. Taxonomy assignment of the representing sequences up to genus level was obtained using a nave Bayesian classifier as implemented in the DADA2 package.