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Differential RNA modifications from dRNA-seq

ABSTRACT: Detection of differential RNA modifications from direct RNA sequencing of human cell lines

PROVIDER: PRJEB40872 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
		Other
	ERR4706160.fastq.gz	Fastqsanger.gz
	ERR4706161.fastq.gz	Fastqsanger.gz
	ERR4708391.fastq.gz	Fastqsanger.gz
	ERR4706162.fastq.gz	Fastqsanger.gz

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Project description:Chemical RNA modifications, collectively referred to as the ‘epitranscriptome’, have been intensively studied during the last years, largely facilitated by the use of next-generation sequencing technologies. Recent efforts have turned towards the nanopore direct RNA sequencing (DRS) platform, as it allows simultaneous detection of diverse RNA modification types in full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling ‘errors’ in DRS datasets, m6A modifications produce very modest ‘errors’, limiting the applicability of this approach to sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully-unmodified in vitro synthetic sequences, increase the ‘error’ signal of m6A modifications, leading to enhanced detection of RNA modifications even at lower stoichiometries. We then show that the use of these models enhances the detection of RNA modifications on previously published in vivo human samples, using third-party softwares for the detection of RNA modifications. Moreover, our work provides a novel RNA basecalling model that shows a median accuracy of 97%, compared to previously available RNA basecalling models that show 91% accuracy. Notably, this increase in accuracy does not only lead to improved detection of RNA modifications, but also enhanced mappability of RNA reads, which becomes more evident in the case of short RNA reads (50% increase). Altogether, our work stresses the importance of using fully unmodified RNA sequences for training RNA basecalling models, and how the use of different basecalling models can significantly affect the detection of RNA modifications and read mappability.

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