Pyrosequencing of the 16S rRNA gene from diverse environments has demonstrated that the microbial diversity can be orders of magnitude higher than appreciated by previous selleck chem technologies (10, 11). This review article discusses several aspects of the pyrosequencing technique, including the principles, applications, and significant contribution to the study of the human microbiome, with special emphasis on the oral microbiome. The Sanger sequencing approach The Sanger approach has stood as the gold standard DNA sequencing technique over the last three decades. In brief, the automated Sanger approach comprises the following steps: DNA purification, DNA synthesis, and labeling using the chain termination method with dye-labeled dideoxynucleotides (ddNTPs), capillary electrophoresis, and fluorescence detection.

In the last years, the automated Sanger method has evolved to generate longer sequence reads of up to 800 bases. The outstanding contribution of Sanger sequencing to scientific advances in diverse areas is incalculable. In oral microbiology, numerous studies have used broad-range PCR, followed by cloning, and Sanger sequencing to unravel the microbiota associated with diverse oral sites in healthy (12, 13) and disease conditions, including caries (3, 14�C16), endodontic infections (4, 6, 17�C20), periodontal diseases (1, 5, 21�C23), periimplantitis (24), and halitosis (2, 25). Collectively, these studies complemented the data from previous culture studies to reveal that more than 1,000 different bacterial species-level taxa belonging to 13 phyla colonized the human oral cavity (26).

Six bacterial phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria) contained the huge majority of oral representatives. Not all of these species-level taxa were present in the same mouth at any one time, and a particular individual usually harbored about 100�C200 taxa (27). Whereas some species were common to different oral sites, the majority of species were selective for a particular site (12). Regardless of the site, as much as 50% or more of the detected taxa have been demonstrated to be as-yet-uncultivated bacteria (28). NGS technologies Next-generation DNA sequencing technologies that permit massive sequencing with a much higher throughput than the Sanger method have become recently available. The first commercially available NGS platform was introduced in 2005 and as of then NGS methods have revolutionized the field of genomic GSK-3 analysis (29).