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.