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Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming

Author

Listed:
  • Bertrand Theulot

    (École normale supérieure, Université PSL, CNRS, INSERM
    Collège Doctoral
    100 Washington Square East)

  • Alan Tourancheau

    (École normale supérieure, Université PSL, CNRS, INSERM)

  • Emma Simonin Chavignier

    (École normale supérieure, Université PSL, CNRS, INSERM)

  • Etienne Jean

    (École normale supérieure, Université PSL, CNRS, INSERM)

  • Jean-Michel Arbona

    (École Normale Supérieure de Lyon, CNRS, UMR5239, INSERM, U1293, Université Claude Bernard Lyon 1
    Case 907, Parc Scientifique de Luminy)

  • Benjamin Audit

    (LPENSL, UMR5672)

  • Olivier Hyrien

    (École normale supérieure, Université PSL, CNRS, INSERM)

  • Laurent Lacroix

    (École normale supérieure, Université PSL, CNRS, INSERM)

  • Benoît Tallec

    (École normale supérieure, Université PSL, CNRS, INSERM)

Abstract

Current temporal studies of DNA replication are either low-resolution or require complex cell synchronisation and/or sorting procedures. Here we introduce Nanotiming, a single-molecule, nanopore sequencing-based method producing high-resolution, telomere-to-telomere replication timing (RT) profiles of eukaryotic genomes by interrogating changes in intracellular dTTP concentration during S phase through competition with its analogue bromodeoxyuridine triphosphate (BrdUTP) for incorporation into replicating DNA. This solely demands the labelling of asynchronously growing cells with an innocuous dose of BrdU during one doubling time followed by BrdU quantification along nanopore reads. We demonstrate in S. cerevisiae model eukaryote that Nanotiming reproduces RT profiles generated by reference methods both in wild-type and mutant cells inactivated for known RT determinants. Nanotiming is simple, accurate, inexpensive, amenable to large-scale analyses, and has the unique ability to access RT of individual telomeres, revealing that Rif1 iconic telomere regulator selectively delays replication of telomeres associated with specific subtelomeric elements.

Suggested Citation

  • Bertrand Theulot & Alan Tourancheau & Emma Simonin Chavignier & Etienne Jean & Jean-Michel Arbona & Benjamin Audit & Olivier Hyrien & Laurent Lacroix & Benoît Tallec, 2025. "Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-024-55520-3
    DOI: 10.1038/s41467-024-55520-3
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    References listed on IDEAS

    as
    1. Santiago E. Sanchez & Yuchao Gu & Yan Wang & Anudeep Golla & Annika Martin & William Shomali & Dirk Hockemeyer & Sharon A. Savage & Steven E. Artandi, 2024. "Digital telomere measurement by long-read sequencing distinguishes healthy aging from disease," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    2. Tobias T. Schmidt & Carly Tyer & Preeyesh Rughani & Candy Haggblom & Jeffrey R. Jones & Xiaoguang Dai & Kelly A. Frazer & Fred H. Gage & Sissel Juul & Scott Hickey & Jan Karlseder, 2024. "High resolution long-read telomere sequencing reveals dynamic mechanisms in aging and cancer," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    3. Riham Smoom & Catherine Lee May & Vivian Ortiz & Mark Tigue & Hannah M. Kolev & Melissa Rowe & Yitzhak Reizel & Ashleigh Morgan & Nachshon Egyes & Dan Lichtental & Emmanuel Skordalakes & Klaus H. Kaes, 2023. "Telomouse—a mouse model with human-length telomeres generated by a single amino acid change in RTEL1," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    4. Bertrand Theulot & Laurent Lacroix & Jean-Michel Arbona & Gael A. Millot & Etienne Jean & Corinne Cruaud & Jade Pellet & Florence Proux & Magali Hennion & Stefan Engelen & Arnaud Lemainque & Benjamin , 2022. "Genome-wide mapping of individual replication fork velocities using nanopore sequencing," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
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