PT  - JOURNAL ARTICLE
AU  - Watson, Kaylee J
AU  - Bromley, Robin E
AU  - Sparklin, Benjamin C
AU  - Gasser, Mark T
AU  - Bhattacharya, Tamanash
AU  - Lebov, Jarrett F
AU  - Tyson, Tyonna
AU  - Dai, Nan
AU  - Teigen, Laura E
AU  - Graf, Karen T
AU  - Foster, Jeremy M
AU  - Michalski, Michelle
AU  - Bruno, Vincent M
AU  - Lindsey, Amelia RI
AU  - Corrêa, Ivan R
AU  - Hardy, Richard W
AU  - Newton, Irene LG
AU  - Dunning Hotopp, Julie C
TI  - Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m<sup>5</sup>C at GCU motifs
AID  - 10.26508/lsa.202302201
DP  - 2024 Feb 01
TA  - Life Science Alliance
PG  - e202302201
VI  - 7
IP  - 2
4099  - http://www.life-science-alliance.org/content/7/2/e202302201.short
4100  - http://www.life-science-alliance.org/content/7/2/e202302201.full
SO  - Life Sci. Alliance2024 Feb 01; 7
AB  - 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 frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.