Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5 C at GCU motifs

Kaylee J. Watson, Robin E. Bromley, Benjamin C. Sparklin, Mark T. Gasser, Tamanash Bhattacharya, Jarrett F. Lebov, Tyonna Tyson, Nan Dai, Laura E. Teigen, Karen T. Graf, Jeremy M. Foster, Michelle Michalski, Vincent M. Bruno, Amelia R.I. Lindsey, Ivan R. Corrêa, Richard W. Hardy, Irene L.G. Newton, Julie C. Dunning Hotopp

Research output: Contribution to journalArticlepeer-review


RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an “Alternative Model” to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a fre-quent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be recon-sidered, including those from human coronavirus and human cerebral organoid samples.

Original languageEnglish (US)
Article numbere202302201
JournalLife science alliance
Issue number2
StatePublished - Feb 2024
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2023 Watson et al.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.


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