Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at...

RNA modifications, such as méthylation, 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 virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.