- The 16S ribosomal RNA (rRNA) gene has long been a fundamental tool in bacterial taxonomy due to its universal presence, conserved structure, and the presence of hypervariable regions that enable phylogenetic comparisons. It has been especially useful for genus-level identification and for characterizing microbial communities through culture-independent approaches.
- However, the resolution of 16S rRNA sequencing is limited, particularly at the species and subspecies levels. Many closely related bacterial species share over 99% sequence identity in their 16S rRNA genes, making it difficult—if not impossible—to distinguish between them using this method alone. Additionally, some bacterial genomes harbor multiple copies of the 16S rRNA gene that may differ slightly, introducing further ambiguity in taxonomic assignment. The relatively slow rate of evolution of this gene also limits its ability to reflect finer phylogenetic distinctions, especially among recently diverged taxa.
- Furthermore, the accuracy of species-level identification using 16S rRNA sequencing is highly dependent on the quality and completeness of reference databases. Misannotations, incomplete entries, or lack of representative sequences can lead to incorrect or uncertain classifications. This is particularly problematic in diverse or poorly characterized bacterial genera, where many environmental isolates remain only partially identified.
- Because of these limitations, reliance solely on 16S rRNA sequencing for bacterial taxonomy can lead to misclassification, underestimation of diversity, or failure to resolve functionally distinct lineages. In the context of modern microbial research—where precise identification is often critical for understanding ecological function, pathogenicity, or probiotic potential—these shortcomings are increasingly apparent.
- To address these challenges, bacterial systematics has entered a new era driven by whole-genome sequencing (WGS), which offers significantly higher taxonomic resolution and functional insight.
