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

Zhihao Guo; Yanwen Shao; Lu Tan; Beifang Lu; Xin Deng; Sheng Chen; Runsheng Li

doi:10.1016/j.crmeth.2025.101168

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

Zhihao Guo (Co-first Author), Yanwen Shao (Co-first Author), Lu Tan, Beifang Lu, Xin Deng, Sheng Chen, Runsheng Li^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

3 Citations (Scopus)

10 Downloads (CityUHK Scholars)

Abstract

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.

© 2025 The Author(s).

Original language	English
Article number	101168
Number of pages	21
Journal	Cell Reports Methods
Volume	5
Issue number	9
Online published	9 Sept 2025
DOIs	https://doi.org/10.1016/j.crmeth.2025.101168
Publication status	Published - 15 Sept 2025

Funding

This work was supported by the Early Career Scheme (CityU 21100521) and General Research Fund (11105524) from the Research Grants Council of the Hong Kong Special Administrative Region, China; the Hong Kong Health and Medical Research Fund (project number 08194126); and new Research Initiatives support from City University of Hong Kong (project number 9610497) to R.L.

Research Keywords

bacterial epitranscriptome
direct RNA sequencing
RNA modification
nanopore sequencing
current comparison tool
modification detection

Publisher's Copyright Statement

This full text is made available under CC-BY 4.0. https://creativecommons.org/licenses/by/4.0/

RGC Funding Information

RGC-funded

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10.1016/j.crmeth.2025.101168

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1 Finished
2 Active

GRF: Locate the Hybrid Male Sterility Related Genes in Two Closely Related Nematodes with an X-Linked Introgression Strain
LI, R. (Principal Investigator / Project Coordinator)
1/01/25 → …
Project: Research
HMRF: Subtyping CN-AML with Modification Biomarkers Using Nanopore Sequencing
LI, R. (Principal Investigator / Project Coordinator) & Leung, A. Y. H. (Co-Investigator)
1/11/21 → …
Project: Research
ECS: Towards A Better Understanding of rDNA in Caenorhabditis Species Using Long-Read Sequencing
LI, R. (Principal Investigator / Project Coordinator)
1/01/22 → 22/12/25
Project: Research

Cite this

@article{94e46f401a6d48408fe64fddd135664a,

title = "Enhanced detection of RNA modifications in Escherichia coli utilizing direct RNA sequencing",

abstract = "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.{\textcopyright} 2025 The Author(s).",

keywords = "bacterial epitranscriptome, direct RNA sequencing, RNA modification, nanopore sequencing, current comparison tool, modification detection",

author = "Zhihao Guo and Yanwen Shao and Lu Tan and Beifang Lu and Xin Deng and Sheng Chen and Runsheng Li",

year = "2025",

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doi = "10.1016/j.crmeth.2025.101168",

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T1 - Enhanced detection of RNA modifications in Escherichia coli utilizing direct RNA sequencing

AU - Guo, Zhihao

AU - Shao, Yanwen

AU - Tan, Lu

AU - Lu, Beifang

AU - Deng, Xin

AU - Chen, Sheng

AU - Li, Runsheng

PY - 2025/9/15

Y1 - 2025/9/15

N2 - 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.© 2025 The Author(s).

AB - 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.© 2025 The Author(s).

KW - bacterial epitranscriptome

KW - direct RNA sequencing

KW - RNA modification

KW - nanopore sequencing

KW - current comparison tool

KW - modification detection

U2 - 10.1016/j.crmeth.2025.101168

DO - 10.1016/j.crmeth.2025.101168

M3 - RGC 21 - Publication in refereed journal

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VL - 5

JO - Cell Reports Methods

JF - Cell Reports Methods

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ER -

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

Abstract

Funding

Research Keywords

Publisher's Copyright Statement

RGC Funding Information

Access Output

Other Access Options

Permanent Link

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Projects

GRF: Locate the Hybrid Male Sterility Related Genes in Two Closely Related Nematodes with an X-Linked Introgression Strain

HMRF: Subtyping CN-AML with Modification Biomarkers Using Nanopore Sequencing

ECS: Towards A Better Understanding of rDNA in Caenorhabditis Species Using Long-Read Sequencing

Cite this