Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing

Mostra el registre complet Registre parcial de l'ítem

  • dc.contributor.author Begik, Oguzhan
  • dc.contributor.author Diensthuber, Gregor
  • dc.contributor.author Liu, Huanle
  • dc.contributor.author Delgado-Tejedor, Anna
  • dc.contributor.author Kontur, Cassandra
  • dc.contributor.author Niazi, Adnan Muhammad
  • dc.contributor.author Valen, Eivind
  • dc.contributor.author Giraldez, Antonio J.
  • dc.contributor.author Beaudoin, Jean-Denis
  • dc.contributor.author Mattick, John S.
  • dc.contributor.author Novoa, Eva Maria
  • dc.date.accessioned 2023-02-24T07:00:46Z
  • dc.date.available 2023-02-24T07:00:46Z
  • dc.date.issued 2023
  • dc.description.abstract RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
  • dc.description.sponsorship We thank all the members of the Novoa lab for their valuable insights and discussion. O.B. is supported by a UNSW International PhD fellowship and Australian Government Research Training Program Scholarship. G.D. is part of the ROPES ITN, which received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 956810. A.D.-T. is supported by an FPI-SO fellowship (PRE2019-088498). This work was supported by the Australian Research Council (DP180103571 to E.M.N.), the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PGC2018-098152-A-100 to E.M.N.), the European Research Council (ERC-StG-2021 No 101042103 to E.M.N.) and the NIH (R35GM122580 and RO1HD00035 to A.J.G). We acknowledge the support of the MEIC to the EMBL partnership, Centro de Excelencia Severo Ochoa and CERCA Programme/Generalitat de Catalunya. We thank T. Mercer (Garvan Institute, Australia) for providing us with the sequins used as spike-ins in Nano3P-seq zebrafish runs. We thank the Sdelci lab (CRG, Spain) for providing us with HeLa cell lines pellets used in this work.
  • dc.format.mimetype application/pdf
  • dc.identifier.citation Begik O, Diensthuber G, Liu H, Delgado-Tejedor A, Kontur C, Niazi AM, Valen E, Giraldez AJ, Beaudoin JD, Mattick JS, Novoa EM. Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing. Nat Methods. 2023 Jan;20(1):75-85. DOI: 10.1038/s41592-022-01714-w
  • dc.identifier.doi http://dx.doi.org/10.1038/s41592-022-01714-w
  • dc.identifier.issn 1548-7091
  • dc.identifier.uri http://hdl.handle.net/10230/55903
  • dc.language.iso eng
  • dc.publisher Nature Research
  • dc.relation.ispartof Nat Methods. 2023 Jan;20(1):75-85
  • dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/956810
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/PGC2018-098152-A-100
  • dc.rights © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
  • dc.rights.accessRights info:eu-repo/semantics/openAccess
  • dc.rights.uri http://creativecommons.org/licenses/by/4.0/
  • dc.subject.keyword RNA metabolism
  • dc.subject.keyword RNA sequencing
  • dc.subject.keyword Transcriptomics
  • dc.title Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing
  • dc.type info:eu-repo/semantics/article
  • dc.type.version info:eu-repo/semantics/publishedVersion

Col·leccions

Articles (Center for Genomic Regulation (CRG))
Articles (Departament de Medicina i Ciències de la Vida)
Documents OpenAIRE (Open Access Infrastructure for Research in Europe)