The composition of subgingival microbiota is related to the status of periodontal health. 16S rRNA sequencing has been proved useful and efficient to assess the composition of subgingival microbiota. However, different homogenization methods utilized to isolate bacterial DNA from subgingival plaque can affect sequencing results, especially from small samples. The study was aimed to compare two common homogenization methods for DNA extraction from plaque samples that allow for accurate genomic sequencing of subgingival microbiota.
Materials and Methods:
Subgingival plaque samples were collected from interproximal sites of molars of one subject and stored in 150 μl TE buffer at -80 0C. Microbial genomic DNA was extracted using a MO BIO Powersoil DNA Isolation Kit. Cell lysis and homogenization was either performed with 0.1 mm Silica Beads (SI) or 0.7 mm Garnet Beads (GA) on a Vortex. 16S rRNA sequencing (Illumina MiSeq) was then performed to create the corresponding subgingival bacterial genomic profile. Taxonomic assignments to operational taxonomic units (OTUs) from phylum to species level were completed using CLC Genomics Workbench v10 with 98% matching to reference databases (Human Oral Microbiome Database).
Primary commensal and periodontal bacterial species including Camphylobacter gracilis, Corynebacterium matruchotii, Fusobacterium nucleatum, and Porphyromonas gingivalis with relative abundance equal to or more than 1% were identified by sequencing in two groups. There was no significant difference of relative abundance in any species between two groups based on homogenization methods. The alpha diversity (Shannon index, OTU numbers) and beta diversity (Bray-Curtis distance) between two groups were not significantly different. Part of the samples in the SI group could not be sequenced due to failure of amplification.
Subgingival bacteria were successfully identified by two bead-based homogenization methods. There was no difference of microbial composition and diversity using different homogenization methods.