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Nanopore Direct RNA Sequencing Data Processing and Analysis Using MasterOfPores

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Computational Epigenomics and Epitranscriptomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2624))

Abstract

This chapter describes MasterOfPores v.2 (MoP2), an open-source suite of pipelines for processing and analyzing direct RNA Oxford Nanopore sequencing data. The MoP2 relies on the Nextflow DSL2 framework and Linux containers, thus enabling reproducible data analysis in transcriptomic and epitranscriptomic studies. We introduce the key concepts of MoP2 and provide a step-by-step fully reproducible and complete example of how to use the workflow for the analysis of S. cerevisiae total RNA samples sequenced using MinION flowcells. The workflow starts with the pre-processing of raw FAST5 files, which includes basecalling, read quality control, demultiplexing, filtering, mapping, estimation of per-gene/transcript abundances, and transcriptome assembly, with support of the GPU computing for the basecalling and read demultiplexing steps. The secondary analyses of the workflow focus on the estimation of RNA poly(A) tail lengths and the identification of RNA modifications. The MoP2 code is available at https://github.com/biocorecrg/MOP2 and is distributed under the MIT license.

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Acknowledgments

This work was partly supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PID2021-128193NB-100 to EMN) and the European Research Council (ERC-StG-2021 No 101042103 to EMN). AD-T is supported by a Severo Ochoa FPI PhD fellowship from the MEIC (PRE2019-088498). We acknowledge the support of the MEIC to the EMBL partnership, Centro de Excelencia Severo Ochoa and CERCA Programme/Generalitat de Catalunya.

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Author notes

  1. Authors Luca Cozzuto and Anna Delgado-Tejedor have equally contributed to this chapter.

Authors and Affiliations

  1. Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain

    Luca Cozzuto, Anna Delgado-Tejedor, Toni Hermoso Pulido, Eva Maria Novoa & Julia Ponomarenko

  2. Universitat Pompeu Fabra (UPF), Barcelona, Spain

    Anna Delgado-Tejedor, Eva Maria Novoa & Julia Ponomarenko

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  1. Luca Cozzuto
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  2. Anna Delgado-Tejedor
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  3. Toni Hermoso Pulido
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  4. Eva Maria Novoa
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  5. Julia Ponomarenko
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Corresponding authors

Correspondence to Eva Maria Novoa or Julia Ponomarenko .

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  1. Genoscope, Évry, France

    Pedro H. Oliveira

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Cozzuto, L., Delgado-Tejedor, A., Hermoso Pulido, T., Novoa, E.M., Ponomarenko, J. (2023). Nanopore Direct RNA Sequencing Data Processing and Analysis Using MasterOfPores. In: Oliveira, P.H. (eds) Computational Epigenomics and Epitranscriptomics. Methods in Molecular Biology, vol 2624. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2962-8_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2962-8_13

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