Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m 5 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 (m 5 C) 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 m 5 C at the central position of a GCU motif. However, it also identified a m 5 C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m 5 C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.