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Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution

Nature Protocols volume 20, pages 3607–3628 (2025)Cite this article

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Abstract

RNA polyadenylation is crucial for RNA maturation, stability and function, with poly(A) tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3′ end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances and tail-composition and tail-length estimates at single-molecule resolution. Taking advantage of a template switching–based protocol, Nano3P-seq can sequence any RNA-derived molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flow cells, as well as compatible software for poly(A) tail length and content prediction, which we term ‘PolyTailor’. We demonstrate that PolyTailor provides accurate estimates of transcript abundances and tail lengths and composition, while capturing both coding and noncoding RNA biotypes, including mRNAs, small nucleolar RNAs and ribosomal RNAs. Nano3P-seq can be applied to RNA samples prepared by using different methods (e.g., poly(A)-selected, ribodepleted or total RNA) and can be completed in 1 day. The protocol requires experience in molecular biology techniques and nanopore sequencing library preparation and basic knowledge of Linux Bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and noncoding transcriptomes in a comprehensive and reproducible manner.

Key points

  • Nano3P-seq uses nanopore-based cDNA sequencing to characterize multiple coding and noncoding RNA biotypes from RNA prepared by using various methods, including poly(A)-selected, ribodepleted or total RNA.

  • The protocol is compatible with the latest R10 sequencing chemistry from Oxford Nanopore Technologies and provides a complementary software package, PolyTailor, to estimate RNA abundance and tail length and composition at the single-molecule level.

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Fig. 1: Schematic overview of the Nano3P-seq library-preparation workflow.
Fig. 2: Applications of the Nano3P-seq protocol.
Fig. 3: Flowchart of Nano3P-seq data-analysis steps.
Fig. 4: Benchmarking the updated Nano3P-seq protocol and PolyTailor.
Fig. 5: Typical RNA and cDNA profile.
Fig. 6: Anticipated results from data analysis.

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Data availability

The data used in this protocol have been deposited in the European Nucleotide Archive at the European Molecular Biology Laboratory-European Bioinformatics Institute under accession number PRJEB80101. A subset of the Nano3P-seq reads used in this manuscript (and their respective reference files) can be downloaded from the Novoa Lab’s public repository for pipeline testing at https://public-docs.crg.es/enovoa/public/lpryszcz/src/polyTailor/test/.

Code availability

The PolyTailor software used for Nano3P-seq analysis, including test data, has been made publicly available in GitHub at https://github.com/novoalab/polyTailor32.

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Acknowledgements

This work was supported by the Spanish Ministry of Science, Innovation and Universities (MCIN/AEI/10.13039/501100011033/FEDER, UE) (PID2021-128193NB-100 to E.M.N.). This project received funding from the European Union’s Horizon Europe under the grant agreement No. 101042103 (to E.M.N.). The views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for these opinions. O.B. was supported by CRG Proof-Of-Concept funding. We acknowledge support from the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa (CEX2020-001049-S, MCIN/AEI /10.13039/501100011033), the Generalitat de Catalunya through the CERCA programme and the EMBL partnership. We are grateful to the CRG Core Technologies Programme for their support and assistance in this work. Figures were drawn by using BioRender.

Author information

Author notes

  1. These authors contributed equally: Oguzhan Begik, Leszek P. Pryszcz.

Authors and Affiliations

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

    Oguzhan Begik, Leszek P. Pryszcz & Eva Maria Novoa

  2. Computational Biology Unit, Department of Informatics, University of Bergen, Bergen, Norway

    Adnan Muhammad Niazi & Eivind Valen

  3. Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway

    Eivind Valen

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

    Eva Maria Novoa

  5. ICREA, Barcelona, Spain

    Eva Maria Novoa

Authors
  1. Oguzhan Begik
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  2. Leszek P. Pryszcz
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  3. Adnan Muhammad Niazi
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  4. Eivind Valen
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  5. Eva Maria Novoa
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Contributions

O.B. adapted the experimental protocol for Nano3P-seq for R10 flow cells. L.P.P. developed the PolyTailor software. O.B., A.M.N., E.V. and E.M.N. troubleshooted the tail-length prediction tool development. O.B. built the figures. E.M.N. supervised the project. O.B., L.P. and E.M.N. wrote the manuscript.

Corresponding author

Correspondence to Eva Maria Novoa.

Ethics declarations

Competing interests

E.M.N. is a member of the Scientific Advisory Board of IMMAGINA Biotech. O.B. and E.M.N. have received travel bursaries from ONT to present their work at conferences.

Peer review

Peer review information

Nature Protocols thanks Bin Tian, Falong Lu, Jingwen Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Begik, O. et al. Nat. Methods 20, 75–85 (2023): https://doi.org/10.1038/s41592-022-01714-w

Delgado-Tejedor, A. et al. Nat. Commun. 15, 10054 (2024): https://doi.org/10.1038/s41467-024-54368-x

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Begik, O., Pryszcz, L.P., Niazi, A.M. et al. Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution. Nat Protoc 20, 3607–3628 (2025). https://doi.org/10.1038/s41596-025-01205-0

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