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Acera Mateos, Pablo ; Sethi, Aditya J. ; Ravindran, Agin ; Guarnacci, Marco ; Srivastava, Akanksha ; Xu, Jiajia ; Woodward, Katrina ; Yuen, Zaka Wing Sze ; Hamilton, William ; Gao, Jing ; Starrs, Lora M. ; Hayashi, Rippei ; Wickramasinghe, Vihandha ; Preiss, Thomas ; Burgio, Gaetan ; Dehorter, Nathalie ; Shirokikh, Nikolay ; Eyras Jiménez, Eduardo Angel
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions, using signals from nanopore direct RNA sequencing. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
Acera Mateos, Pablo ; Sethi, Aditya J. ; Guarnacci, Marco ; Ravindran, Agin ; Srivastava, Akanksha ; Xu, Jiajia ; Woodward, Katrina ; Hamilton, William ; Gao, Jing ; Starrs, Lora M. ; Burgio, Gaetan ; Hayashi, Rippei ; Wickramasinghe, Vihandha ; Dehorter, Nathalie ; Preiss, Thomas ; Shirokikh, Nikolay ; Eyras Jiménez, Eduardo Angel
The expanding field of epitranscriptomics might rival the epigenome in the diversity of the biological processes impacted. However, the identification of modifications in individual RNA molecules remains challenging. We present CHEUI, a new method that detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) at single-nucleotide and single-molecule resolution from Nanopore signals. CHEUI predicts methylation in Nanopore reads and transcriptomic sites in a single condition, and differential m6A and m5C methylation between any two conditions. Using extensive benchmarking with Nanopore data derived from synthetic and natural RNA, CHEUI showed higher accuracy than other existing methods in detecting m6A and m5C sites and quantifying the site stoichiometry levels, while maintaining a lower proportion of false positives. CHEUI provides a new capability to detect RNA modifications with high accuracy and resolution that can be cost-effectively expanded to other modifications to unveil the full span of the epitranscriptome in normal and disease conditions.
Acera Mateos, Pablo ; Sethi, Aditya J. ; Ravindran, Agin ; Srivastava, Akanksha ; Woodward, Katrina ; Mahmud, Shafi ; Kanchi, Madhu ; Guarnacci, Marco ; Xu, Jiajia ; Yuen, Zaka Wing Sze ; Zhou, You ; Sneddon, Alexandra ; Hamilton, William ; Gao, Jing ; Starrs, Lora M. ; Hayashi, Rippei ; Wickramasinghe, Vihandha ; Zarnack, Katharina ; Preiss, Thomas ; Burgio, Gaetan ; Dehorter, Nathalie ; Shirokikh, Nikolay ; Eyras Jiménez, Eduardo Angel
The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI’s capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.
Acera Mateos, Pablo ; Sethi, Aditya J. ; Guarnacci, Marco ; Ravindran, Agin ; Srivastava, Akanksha ; Xu, Jiajia ; Woodward, Katrina ; Hamilton, William ; Gao, Jing ; Starrs, Lora M. ; Burgio, Gaetan ; Hayashi, Rippei ; Wickramasinghe, Vihandha ; Dehorter, Nathalie ; Preiss, Thomas ; Shirokikh, Nikolay ; Eyras Jiménez, Eduardo Angel
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.