Enhanced detection of RNA modifications in Escherichia coli utilizing direct RNA sequencing

RNA modifications play crucial roles in prokaryotic cellular processes. In this study, we found that the recent advances in direct RNA sequencing have improved yield, accuracy, and signal-to-noise ratio in bacterial samples. By evaluating four current RNA modification calling models in Escherichia coli transcriptome using native and in vitro transcribed (IVT) RNA, we found the models identified most known rRNA modifications but produced false positives. To address this, we developed nanoSundial, a comparative method leveraging raw current signals from native and IVT samples to de novo identify multiple RNA modifications. We optimized nanoSundial on well-studied E. coli rRNA modification sites and validated its effectiveness with tRNAs. It identified 190 stably modified mRNA regions, which enriched near the ends of highly expressed operons. This study highlighted the strengths and limitations of current nanopore-based modification detection methods on bacterial RNA, introduced a robust comparative tool, and elucidated previously uncharacterized mRNA modification landscapes.