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Nanocompore

ABSTRACT: RNA modifications mapping by comparative Nanopore direct RNA sequencing

PROVIDER: PRJEB35148 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	ERR3700282.fastq.gz	Fastqsanger.gz
	ERR3711992.fastq.gz	Fastqsanger.gz
	ERR3711993.fastq.gz	Fastqsanger.gz
	ERR3711994.fastq.gz	Fastqsanger.gz

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Single-Molecule Direct RNA Sequencing Reveals the Shaping of Epitranscriptome Across Multiple Species

Project description:RNA modifications play a crucial role in gene regulation, yet understanding their transcriptome-wide landscape and biogenesis mechanisms remains challenging. Traditional next-generation sequencing (NGS) methods detect RNA modifications based on the aggregation of short-reads, overlooking the nature of RNA as multiple transcripts from one gene. Third-generation sequencing (TGS) platforms provide direct RNA sequencing at the resolution of individual RNA molecules, offering the promise of detecting RNA modifications and RNA processing events simultaneously. In this study, we introduce SingleMod, a deep learning model tailored for precise m6A modification mapping on individual RNA molecules using nanopore direct RNA sequencing (DRS). We systematically dissect the transcriptome-wide m6A landscape at single-molecule and single-base resolution, refining our understanding of the genomics of m6A and revealing an additive mode through which m6A shapes the epitranscriptome. Through comparative analyses across diverse species, we quantitatively elucidate three distinct m6A distribution patterns that suggest diverse biogenesis mechanisms. This study pioneers single-molecule epitranscriptome exploration across multiple species, deepening our understanding of m6A, including its genomics, biogenesis, mechanisms, and biological implications.

2025-04-29 | GSE246632 | GEO

Simultaneous profiling of ac4C and m5C modifications from nanopore direct RNA sequencing

Project description:Long-read nanopore sequencing has emerged as a potent tool for studying RNA modifications. However, the detection of N4-acetylcytidine (ac4C) based on nanopore sequencing remains largely unexplored. Here, we introduce ac4Cnet, a deep learning frame utilizing Oxford Nanopore direct RNA sequencing to accurately identify ac4C sites. Our methodology involves training ac4Cnet capable of distinguishing ac4C from unmodified cytidine and 5-methylcytosine (m5C), as well as estimating the modification rate at each ac4C site. We demonstrate the robustness of our approach through validations on independent in vitro datasets and a human cell line, highlighting its versatility and potential for advancing the study of ac4C modifications.

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Project description:Myelodysplastic syndrome (MDS) is a clonal myeloid neoplasm characterized by ineffective haematopoiesis and cytopenia with frequent epigenetic modifications. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) are standard therapeutic options in MDS, but drug resistance is not uncommon.To study RNA modification in particular M6A, P39-AZA-S and P39-AZA-R native RNA libraries were prepared using the direct RNA sequencing kit (Oxford Nanopore) following the manufacturer’s protocol (SQK-RNA002). Differential methylated RNA signal called from raw fast5 files were used as input for basecalling and downstream mapping and methylated transcript detection.

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The detecting of m6A RNA modifications upon Oxford Nanopore direct-RNA sequencing [Nanopore]

Project description:The detecting of m6A RNA modifications upon Oxford Nanopore direct-RNA sequencing [Nanopore]

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N6-methyladenosine modification is not a general trait of positive-sense RNA viral genomes

Project description:<p>Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m6A in CHIKV and DENV RNAs. For this, we combined m6A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m6A machinery’s localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.</p>

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mRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation

Project description:Pseudouridine (psi) is one of the most abundant human mRNA modifications generated via psi synthases, including TRUB1 and PUS7. Nanopore direct RNA sequencing combined with our recent tool, Mod-p ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting changes in positional psi occupancies across cell types, which can inform our understanding of the impact on gene expression. We sequenced, mapped, and compared the positional psi occupancy across six immortalized human cell lines derived from diverse tissue types. We found that lung-derived cells have the highest proportion of psi, while liver-derived cells have the lowest. Further, we find that conserved psi positions correspond to higher levels of protein expression than expected, suggesting translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. Finally, we characterize sites with multiple psi modifications on the same transcript and found that these can be conserved or cell type specific, including examples of multiple psi modifications within the same k-mer. Our data support the hypothesis that psi modifications contribute to regulating translation and that cell type-specific trans-acting factors play a major role in driving pseudouridylation.

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