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

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.