Enhanced Detection of RNA Modifications in Escherichia coli Utilizing Nanopore RNA004 Technology

RNA modifications are critical regulators of diverse cellular processes, yet their roles in prokaryotic mRNAs remain poorly understood. Recent advances in Oxford Nanopore sequencing—especially the RNA004 kit—have enabled higher yields, reduced signal-to-noise ratio, and improved read accuracy, making them promising tools for investigating bacterial epitranscriptomes. Here, we presented a comprehensive walkthrough for Escherichia coli RNA modification analysis based on RNA004. Using both native (WT) and in vitro-transcribed (IVT) RNA samples, we first evaluated the Dorado modification detection models (Ψ, m⁶A, m⁵C, and A-to-I). While each model successfully identified known rRNA modification sites, it also generated many false positives, emphasizing the need for careful data interpretation.

To address these limitations, we introduced nanoSundial ( https://github.com/lrslab/nanoSundial ), a new comparative method that leveraged raw current features from WT and IVT samples to detect multiple types of RNA modifications in prokaryotes. We optimized nanoSundial on well-studied rRNA sites and validated its effectiveness with tRNA modifications. Through technical and biological replicate analyses, nanoSundial demonstrated reproducibility exceeding 95% in tRNA, rRNA, and ncRNA regions, albeit with lower reproducibility (∼61%) in mRNA. We further found enrichment of mRNA modifications at the start or end of coding sequences. In total, 190 stably modified CDS regions were identified in E. coli , many of which cluster near the end of highly expressed transcriptional units (TUs) in each operon.

Overall, this study highlighted the strengths and limitations of current nanopore-based modification detection methods on bacterial RNA, introduced a robust new comparative tool, and elucidated previously uncharacterized mRNA modification landscapes. Our findings open new avenues for understanding the functional impacts of bacterial RNA modifications and advancing epitranscriptomic research in prokaryotes.