# Oral Microbiome and Host Health: Review on Current Advances in Genome-Wide Analysis
## 3 Methodology of the Microbiome Research  
Completion of the HGP was accompanied by parallel and ongoing development of commercially available high-throughput DNA sequencing tools, which eventually facilitated the acquisition of genomic information from human samples. High-throughput techniques such as NGS have replaced the culture-based traditional methods for identifying and characterizing microbes [15]. Past studies without NGS and relying on the laboratory culture system were limited to small parts, such as the biochemical or physiological properties of microbiomes, called opportunistic pathogens [19]. In the beginning, microbiology research had focused on identifying pathogens within the commensal microbiota and deciphering their virulence in relation to health and disease. In the 1980s, a sequencing technique was introduced that enabled nucleotide sequences to be determined. This remarkable advance provided new and diverse information about microbial taxonomic profiling regardless of cultivability [19]. In taxonomic profiling, two methods, amplicon sequencing (16S rRNA sequencing) and direct shotgun sequencing (metagenomics), are used [19]. In the oral microbiome studies, samples were taken from dental plaque and circular fluid from the periodontal pocket. This technology has allowed the characterization of microbial diversity in the human microbiome with unprecedented depth and coverage [20]. These oral microbiome studies originated from HMP's 16S rRNA sequencing data, published in the Human Oral Microbiome Database (HOMD), which identified oral bacteria from specific locations in the mouth such as the teeth, the gingival sulcus, the tongue, the cheek, the tonsils, and the soft and hard palates [2]. These early sequencing studies focused on the 16S rRNA sequence, and this conventional sequencing method was performed using a cultivated clonal culture; however, there were many microorganisms that were unculturable and thus could not be identified with this conventional method (Figure 2) [21]. This cultivation-based method could find \(<1\%\) of microbial species; therefore, metagenomics was introduced to identify non-isolated organisms via DNA sequencing without the need for cultivation and isolation [21]. In addition to metagenomics, DNA barcoding or metabarcoding is also used for the identification of species, using a short section of DNA from a reference library database [22]. The term \"metabarcoding\" is used when DNA barcoding is used to identify organisms from samples containing DNA from more than one organism [23]. Metagenomics also enables the metabolic and functional diversity of microbial communities to be accessed via metatranscriptomics, metaproteomics, and metabolomics [24]. Metaproteomics has emerged as a complementary approach to identify the functions of microbial communities [25]. Mass spectrometry-based proteomics has been widely used for studying the composition of proteins in oral microbial communities and has been applied to characterize oral biofilms [26]. Many metaproteomic studies have been performed in periodontal disease and endodontic infections associated with apical periodontitis using saliva and crevicular fluids [27]. Metabolomics is the study of metabolites within a biologic system to discover what happens in cells. Several studies have been conducted on the salivary metabolome; however, these have offered less specific explanations due to difficulties regarding disentangling host reactions and microbial contributions [27]. As such, metaproteomics and metabolomics complement the foundations established by metagenomics and metatranscriptomics in the comprehensive understanding of microbiomes.