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. 2023 Nov 29;7(2):e202302201.
doi: 10.26508/lsa.202302201. Print 2024 Feb.

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs

Kaylee J Watson  1 , Robin E Bromley  1 , Benjamin C Sparklin  1 , Mark T Gasser  1 , Tamanash Bhattacharya  2 , Jarrett F Lebov  1 , Tyonna Tyson  1 , Nan Dai  3 , Laura E Teigen  4 , Karen T Graf  1 , Jeremy M Foster  3 , Michelle Michalski  4 , Vincent M Bruno  1   5 , Amelia Ri Lindsey  2 , Ivan R Corrêa Jr  3 , Richard W Hardy  2 , Irene Lg Newton  2 , Julie C Dunning Hotopp  6   5   7
Affiliations

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs

Kaylee J Watson et al. Life Sci Alliance. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1.
Figure 1.. Oxford Nanopore Technologies direct RNA sequencing and GCU motif detection.
(A) Schematic showing how RNA molecules are directly sequenced with Oxford Nanopore Technologies, followed by basecalling of the signal data produced by changes in ionic current, mapping to a reference, and detection of modified bases. Adapted from “Nanopore Sequencing,” by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates. (B) Three most significantly enriched MEME suite motifs in the 10-nucleotide sequences surrounding the top 1,000 putative modifications detected by the Tombo 5-methylcytosine Alternative Model for B. malayi, D. ananassae, C. albicans, E. coli, A549 native RNA, and A549 in vitro transcribed RNA. The top five motifs are shown in Table S3.
Figure S1.
Figure S1.. Histogram of A549 native RNA methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S2.
Figure S2.. Histogram of A549 in vitro transcription RNA methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S3.
Figure S3.. Histogram of B. malayi methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S4.
Figure S4.. Histogram of D. ananassae methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S5.
Figure S5.. Histogram of C. albicans methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S6.
Figure S6.. Histogram of E. coli methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S7.
Figure S7.. Histogram of SARS-CoV-2 native RNA methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S8.
Figure S8.. Histogram of SARS-CoV-2 in vitro transcription RNA methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S9.
Figure S9.. Histogram of curlcake synthetic sequence methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure 2.
Figure 2.. Methylated fractions predicted by the Tombo Alternative Model for 5-methylcytosine.
(A) Density plots of the methylated fraction at all 3-mers containing a central cytosine in native and in vitro transcription viral RNA. Cytosine positions were filtered for depth >10 and methylated fraction >0 in both in vitro transcription and native samples. Histograms are available in Figs S10 and S11. (B) Boxplot showing distributions of the methylated fractions detected by the Tombo 5-methylcytosine Alternative Model. The methylated fraction was extracted for cytosines with a depth >100 except in the lower depth Sindbis virus samples, where cytosines with a depth >10 were retained. Methylated fractions were grouped based on the non-GCU and GCU sequence context. Statistical significance based on P-value from a two-tailed Z test and Cohen’s d effect size.
Figure S10.
Figure S10.. Histogram of Sindbis virus native RNA methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
Figure S11.
Figure S11.. Histogram of Sindbis virus in vitro transcription RNA methylated fractions detected by the Tombo Alternative Model.
Results plotted with R v4.0.3 using a bin width of 0.025.
See this image and copyright information in PMC

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