High-accuracy hierarchical rRNA operon profiling resolves genomovar-level taxonomy and microdiversity using Nanopore sequencing.

Genomovar-level and intragenomic diversity cannot be resolved by conventional amplicon sequencing due to the limitation of fragment lengths and read accuracy, while the application of metagenomic profiling to a large number of samples can be resource intensive. Here, we report UltraRes-rrn, an integrated wet-lab and computational workflow for high-accuracy rrn (i.e., 16S-ITS-23S rRNA) operon profiling using Nanopore sequencing. By integrating ultra-long DNA recovery, long-read amplicon sequencing, and unique molecular identifiers (UMI)-based consensus correction, UltraRes-rrn obtains full-length 16S-ITS-23S rRNA operon consensus sequences with mean accuracies exceeding 99.98%. To achieve higher resolution, we propose a hierarchical rRNA operon profiling strategy in which concatenated 16S+23S rRNA genes (16S23S) serve as a primary and the internal transcribed spacer (ITS) provides a secondary marker. The 16S23S marker achieved discrimination at the genomovar level compared to either 16S or 23S rRNA genes alone, which mitigates the ITS-driven over-splitting observed with the full-length rrn operon and allows for larger proportion of data being classified at higher confidence thresholds. Further, ITS variation was strongly structured by tRNA occurrence patterns, suggesting that ITS can capture taxon microdiversity missed by either 16S or 23S rRNA gene sequences alone. The UltraRes-rrn workflow was applied to full-scale nitrogen removal reactors, revealing intraspecies diversity variation driven by different carbon regimes, which would not have been possible with a shorter gene sequence. In summary, UltraRes-rrn enables cost-effective community profiling at genomovar-level resolution in complex ecosystems.