In Silico and Experimental Assessment of the 16S-ITS-23S Operon as a Genetic Marker for Bacterial Species Identification

High-accuracy long-read sequencing of the 16S-ITS-23S rRNA operon offers a powerful tool for bacterial taxonomic classification. We evaluated its resolving power against both full-length 16S rRNA and hypervariable regions (HVRs) using a dual in silico and experimental framework with rat faecal samples. The operon demonstrated superior accuracy in both analyses. In silico, it yielded the highest proportion of pure species clusters (96.8%), significantly outperforming full-length 16S (80.5%) and HVRs (<66%). Experimentally, it enabled species-level assignment for 75.2% of sequences, outperforming full-length 16S (62.1%) and the standard V4 HVR (18.5%). An internal bias-control analysis confirmed these differences were due to the superior information content of the operon marker itself. Our findings provide robust evidence that sequencing the 16S-ITS-23S operon is a superior method for culture-free bacterial profiling, and offers a new gold standard for high-resolution taxonomic profiling in complex environmental and host-associated samples.