Oral Bacteria DNA Extraction and Sequencing

Genomic DNA from oral bacteria was extracted from oral wash samples using the MoBio PowerSoil DNA Isolation Kit (Qiagen). 16Sv3-4 rRNA gene sequencing was performed on the extracted bacterial DNA and gene amplicon libraries were generated as reported previously (15, 43 (link)). Briefly, libraries were created to allow for sequencing covering variable regions V3 to V4 (Primers: 347F-5′GGAGGCAGCAGTAAGGAAT-3′ and 803R- 5′CTACCGGGGTATCTAATCC-3′). For 16Sv3-4 rRNA gene amplification preparation, 5 ng of genomic DNA was used as the template in 25 μL of PCR reaction buffer. The PCR amplicons were then purified using the Agencourt AMPure XP kit (Beckman Coulter) and purified by fluorometry using the Quant-I T PicoGreen dsDNA Assay Kit (Invitrogen; ref. 15 (link)). A total of 10⁷ molecules/μL of purified amplicons were pooled for sequencing using Roche 454 GS FLX Titanium pyrosequencing system.