16S

16S commonly refers to the 16S ribosomal RNA gene, a component of the 30S small subunit of prokaryotic ribosomes. It is highly conserved across bacteria and archaea, yet contains variable regions that allow discrimination among taxa. The gene is typically about 1,500 base pairs in length and is organized within the rRNA operon. Within the gene are nine hypervariable regions (V1–V9) separated by conserved sequence blocks, which serve as primer binding sites for PCR amplification.

Because of its combination of conserved and variable regions, the 16S gene is used as a universal phylogenetic marker. It enables identification andClassification of microorganisms, especially when culture is not feasible. In practice, most studies sequence a portion of the gene, often one or several hypervariable regions (for example V3–V4), using PCR primers and high-throughput sequencing. Full-length 16S can be obtained by Sanger sequencing but is less common in large surveys.

Data analysis involves matching sequence reads to reference databases such as SILVA, Greengenes, or RDP, and grouping similar sequences into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) for downstream analyses of community composition and diversity (alpha and beta diversity).

Limitations include limited resolution at the species level, PCR and primer biases, unequal gene copy number among genomes, and inability to differentiate functionally distinct but closely related taxa. The technique also cannot distinguish live from dead cells and may misrepresent taxa with multiple operons or intragenomic heterogeneity. Nevertheless, 16S rRNA gene sequencing remains a foundational method in microbiology and environmental microbiology.