The study evaluates the potential of 16S rRNA gene sequencing for species and strain-level microbiome analysis. The authors use in silico and sequence-based experiments to demonstrate that targeting short regions of the 16S gene with short-read sequencing platforms cannot achieve the taxonomic resolution provided by full-length sequencing. Full-length sequencing platforms are shown to be accurate enough to resolve subtle nucleotide substitutions between intragenomic copies of the 16S gene, indicating that modern analysis approaches must account for intragenomic variation. The study also highlights the prevalence of intragenomic 16S gene copy variants in the human gut microbiome, suggesting their potential to improve taxonomic resolution at species and strain levels. The results support the use of full-length 16S rRNA gene sequencing for high-resolution taxonomic analysis in microbiome studies.The study evaluates the potential of 16S rRNA gene sequencing for species and strain-level microbiome analysis. The authors use in silico and sequence-based experiments to demonstrate that targeting short regions of the 16S gene with short-read sequencing platforms cannot achieve the taxonomic resolution provided by full-length sequencing. Full-length sequencing platforms are shown to be accurate enough to resolve subtle nucleotide substitutions between intragenomic copies of the 16S gene, indicating that modern analysis approaches must account for intragenomic variation. The study also highlights the prevalence of intragenomic 16S gene copy variants in the human gut microbiome, suggesting their potential to improve taxonomic resolution at species and strain levels. The results support the use of full-length 16S rRNA gene sequencing for high-resolution taxonomic analysis in microbiome studies.