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

The detection of chemical modifications to RNA is a rapidly expanding field within epigenetics. Nanopore sequencing technology provides an attractive means of detecting these modifications directly on the RNA, but accurate modification predictions are dependent upon the software developed to interpret the sequencing results. One of these tools, Tombo, allows users to detect modifications using sequencing results from a single RNA sample. However, we find that this method falsely predicts modifications in a specific sequence context across a variety of RNA samples, including RNA that lacks modifications. Results from previous publications include predictions in human coronaviruses with this sequence context and should be reconsidered. Our results highlight the importance of using RNA modification detection tools with caution in the absence of a control RNA sample for comparison.