UCLA

Metagenomics using high-throughput DNA sequencing technologies is becoming a common approach for microbial community characterization. However, not many studies have compared the traditional 16S rRNA clone library approach with the new metagenomic approaches. In this study, we analyzed the community structure of the subgingival microbiome of periodontitis before and after initial therapy. Two approaches were employed in parallel - metagenomic shotgun sequencing and the traditional 16S rRNA clone library method using Sanger sequencing. This allowed us to compare the two approaches. DNA was extracted from subgingival plaque samples obtained from sites with chronic periodontitis in four systemically healthy subjects prior to and 4-6 weeks following initial therapy. For metagenomics sequencing, 100 bp paired-end reads were generated using Illumina sequencing platforms. Two analysis methods were used in analyzing the metagenomic sequencing data. First, the 16S rRNA reads were extracted and aligned against SILVA rRNA database for taxonomic assignment. Second, the metagenomic reads were mapped against a database of microbial reference genomes for genome identification. For clone library analysis, the 16S rRNA gene was amplified and cloned. Plasmid inserts were sequenced bidirectionally and the taxa were assigned using the Microbiome Utilities Portal of Broad Institute. Statistical evaluation of the results demonstrated that the two sequencing approaches revealed consistent microbial community structure of the oral microbiome. The three analysis methods based on the metagenomic sequencing data and clone library data uncovered the presence of a diverse bacterial community in the subgingiva of periodontitis with little detection bias of the major oral microorganisms. The three analyses demonstrated consistent overall decreases in Porphyromonas, Neisseria and Treponema, and increases in Fusobacterium, Veillonella, Prevotella and Streptococcus after treatment. We observed microbial community shift from disease to resolution of periodontitis at genus level, which could allow the use of a few microbial markers in periodontitis diagnosis and monitoring.