Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Multiscale analysis of genome-wide replication timing profiles using a wavelet-based signal-processing algorithm

Abstract

In this protocol, we describe the use of the LastWave open-source signal-processing command language (http://perso.ens-lyon.fr/benjamin.audit/LastWave/) for analyzing cellular DNA replication timing profiles. LastWave makes use of a multiscale, wavelet-based signal-processing algorithm that is based on a rigorous theoretical analysis linking timing profiles to fundamental features of the cell's DNA replication program, such as the average replication fork polarity and the difference between replication origin density and termination site density. We describe the flow of signal-processing operations to obtain interactive visual analyses of DNA replication timing profiles. We focus on procedures for exploring the space-scale map of apparent replication speeds to detect peaks in the replication timing profiles that represent preferential replication initiation zones, and for delimiting U-shaped domains in the replication timing profile. In comparison with the generally adopted approach that involves genome segmentation into regions of constant timing separated by timing transition regions, the present protocol enables the recognition of more complex patterns of the spatio-temporal replication program and has a broader range of applications. Completing the full procedure should not take more than 1 h, although learning the basics of the program can take a few hours and achieving full proficiency in the use of the software may take days.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Modeling the spatio-temporal replication program in a single cell.
Figure 2: Multiscale decomposition of the DNA replication timing profile in HeLa cells of a fragment of chromosome 1.
Figure 3: Multiscale detection of peaks in DNA replication timing profiles.
Figure 4: Identifying replication timing U-domains.
Figure 5: Quantitative analysis of replication program parameters.

Similar content being viewed by others

References

  1. Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

    Article  CAS  Google Scholar 

  2. DePamphilis, M.L. (ed). DNA Replication and Human Disease (Cold Spring Harbor Laboratory Press, 2006).

  3. Aladjem, M.I. Replication in context: dynamic regulation of DNA replication patterns in metazoans. Nat. Rev. Genet. 8, 588–600 (2007).

    Article  CAS  Google Scholar 

  4. Hamlin, J.L., Mesner, L.D. & Dijkwel, P.A. A winding road to origin discovery. Chromosome Res. 18, 45–61 (2010).

    Article  CAS  Google Scholar 

  5. Mesner, L.D., Crawford, E.L. & Hamlin, J.L. Isolating apparently pure libraries of replication origins from complex genomes. Mol. Cell 21, 719–726 (2006).

    Article  CAS  Google Scholar 

  6. Lucas, I. et al. High-throughput mapping of origins of replication in human cells. EMBO Rep. 8, 770–777 (2007).

    Article  CAS  Google Scholar 

  7. The ENCODE Project Consortium. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).

  8. Cadoret, J.-C. et al. Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc. Natl. Acad. Sci. USA 105, 15837–15842 (2008).

    Article  CAS  Google Scholar 

  9. Karnani, N., Taylor, C.M. & Dutta, A. Microarray analysis of DNA replication timing. Methods Mol. Biol. 556, 191–203 (2009).

    Article  CAS  Google Scholar 

  10. Cayrou, C. et al. Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features. Genome Res. 21, 1438–1449 (2011).

    Article  CAS  Google Scholar 

  11. Mesner, L.D. et al. Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription. Genome Res. 21, 377–389 (2011).

    Article  CAS  Google Scholar 

  12. Hyrien, O. & Méchali, M. Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. EMBO J. 12, 4511–4520 (1993).

    Article  CAS  Google Scholar 

  13. Hyrien, O., Maric, C. & Méchali, M. Transition in specification of embryonic metazoan DNA replication origins. Science 270, 994–997 (1995).

    Article  CAS  Google Scholar 

  14. Gerbi, S.A. & Bielinsky, A.K. DNA replication and chromatin. Curr. Opin. Genet. Dev. 12, 243–248 (2002).

    Article  CAS  Google Scholar 

  15. Schübeler, D. et al. Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing. Nat. Genet. 32, 438–442 (2002).

    Article  Google Scholar 

  16. Anglana, M., Apiou, F., Bensimon, A. & Debatisse, M. Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing. Cell 114, 385–394 (2003).

    Article  CAS  Google Scholar 

  17. Fisher, D. & Méchali, M. Vertebrate HoxB gene expression requires DNA replication. EMBO J. 22, 3737–3748 (2003).

    Article  CAS  Google Scholar 

  18. Gilbert, D.M. Making sense of eukaryotic DNA replication origins. Science 294, 96–100 (2001).

    Article  CAS  Google Scholar 

  19. MacAlpine, D.M. & Bell, S.P. A genomic view of eukaryotic DNA replication. Chromosome Res. 13, 309–326 (2005).

    Article  CAS  Google Scholar 

  20. Méchali, M. Eukaryotic DNA replication origins: many choices for appropriate answers. Nat. Rev. Mol. Cell Biol. 11, 728–738 (2010).

    Article  Google Scholar 

  21. Gilbert, D.M. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat. Rev. Genet. 11, 673–684 (2010).

    Article  CAS  Google Scholar 

  22. Ryba, T., Battaglia, D., Pope, B.D., Hiratani, I. & Gilbert, D.M. Genome-scale analysis of replication timing: from bench to bioinformatics. Nat. Protoc. 6, 870–895 (2011).

    Article  CAS  Google Scholar 

  23. Raghuraman, M.K. et al. Replication dynamics of the yeast genome. Science 294, 115–121 (2001).

    Article  CAS  Google Scholar 

  24. MacAlpine, D.M., Rodriguez, H.K. & Bell, S.P. Coordination of replication and transcription along a Drosophila chromosome. Genes Dev. 18, 3094–3105 (2004).

    Article  CAS  Google Scholar 

  25. Eaton, M.L. et al. Chromatin signatures of the Drosophila replication program. Genome Res. 21, 164–174 (2011).

    Article  CAS  Google Scholar 

  26. Farkash-Amar, S. et al. Global organization of replication time zones of the mouse genome. Genome Res. 18, 1562–1570 (2008).

    Article  CAS  Google Scholar 

  27. Hiratani, I. et al. Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6, e245 (2008).

    Article  Google Scholar 

  28. White, E.J. et al. DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states. Proc. Natl. Acad. Sci. USA 101, 17771–17776 (2004).

    Article  CAS  Google Scholar 

  29. Woodfine, K. et al. Replication timing of the human genome. Hum. Mol. Genet. 13, 191–202 (2004).

    Article  CAS  Google Scholar 

  30. Jeon, Y. et al. Temporal profile of replication of human chromosomes. Proc. Natl. Acad. Sci. USA 102, 6419–6424 (2005).

    Article  CAS  Google Scholar 

  31. Woodfine, K. et al. Replication timing of human chromosome 6. Cell Cycle 4, 172–176 (2005).

    Article  CAS  Google Scholar 

  32. Karnani, N., Taylor, C., Malhotra, A. & Dutta, A. Pan-S replication patterns and chromosomal domains defined by genome-tiling arrays of ENCODE genomic areas. Genome Res. 17, 865–876 (2007).

    Article  CAS  Google Scholar 

  33. Desprat, R. et al. Predictable dynamic program of timing of DNA replication in human cells. Genome Res. 19, 2288–2299 (2009).

    Article  CAS  Google Scholar 

  34. Hansen, R.S. et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc. Natl. Acad. Sci. USA 107, 139–144 (2010).

    Article  CAS  Google Scholar 

  35. Chen, C.-L. et al. Impact of replication timing on non-CpG and CpG substitution rates in mammalian genomes. Genome Res. 4, 447–457 (2010).

    Article  Google Scholar 

  36. Weddington, N. et al. Replicationdomain: a visualization tool and comparative database for genome-wide replication timing data. BMC Bioinformatics 9, 530 (2008).

    Article  Google Scholar 

  37. Guilbaud, G. et al. Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome. PLoS Comput. Biol. 7, e1002322 (2011).

    Article  CAS  Google Scholar 

  38. Ryba, T. et al. Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res. 20, 761–770 (2010).

    Article  CAS  Google Scholar 

  39. Yaffe, E. et al. Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture. PLoS Genet. 6, e1001011 (2010).

    Article  Google Scholar 

  40. Friedman, K.L., Brewer, B.J. & Fangman, W.L. Replication profile of Saccharomyces cerevisiae chromosome VI. Genes Cells 2, 667–678 (1997).

    Article  CAS  Google Scholar 

  41. Patel, P.K., Arcangioli, B., Baker, S.P., Bensimon, A. & Rhind, N. DNA replication origins fire stochastically in fission yeast. Mol. Biol. Cell 17, 308–316 (2006).

    Article  CAS  Google Scholar 

  42. Rhind, N. DNA replication timing: random thoughts about origin firing. Nat. Cell Biol. 8, 1313–1316 (2006).

    Article  CAS  Google Scholar 

  43. Czajkowsky, D.M., Liu, J., Hamlin, J.L. & Shao, Z. DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI. J. Mol. Biol. 375, 12–19 (2008).

    Article  CAS  Google Scholar 

  44. de Moura, A.P.S., Retkute, R., Hawkins, M. & Nieduszynski, C.A. Mathematical modelling of whole chromosome replication. Nucleic Acids Res. 38, 5623–5633 (2010).

    Article  CAS  Google Scholar 

  45. Rhind, N., Yang, S.C. & Bechhoefer, J. Reconciling stochastic origin firing with defined replication timing. Chromosome Res. 18, 35–43 (2010).

    Article  CAS  Google Scholar 

  46. Retkute, R., Nieduszynski, C.A. & de Moura, A. Dynamics of DNA replication in yeast. Phys. Rev. Lett. 107, 068103 (2011).

    Article  Google Scholar 

  47. Yang, S.C., Rhind, N. & Bechhoefer, J. Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing. Mol. Syst. Biol. 6, 404 (2010).

    Article  Google Scholar 

  48. Hyrien, O. & Goldar, A. Mathematical modelling of eukaryotic DNA replication. Chromosome Res. 18, 147–161 (2010).

    Article  CAS  Google Scholar 

  49. Goldar, A., Marsolier-Kergoat, M.-C. & Hyrien, O. Universal temporal profile of replication origin activation in eukaryotes. PLoS ONE 4, e5899 (2009).

    Article  Google Scholar 

  50. Baker, A., Audit, B., Yang, S.C., Bechhoefer, J. & Arneodo, A. Inferring where and when replication initiates from genome-wide replication timing data. Phys. Rev. Lett. 108, 268101 (2012).

    Article  CAS  Google Scholar 

  51. Goldar, A., Labit, H., Marheineke, K. & Hyrien, O. A dynamic stochastic model for DNA replication initiation in early embryos. PLoS ONE 3, e2919 (2008).

    Article  Google Scholar 

  52. Brodie of Brodie, E.-B. et al. From DNA sequence analysis to modeling replication in the human genome. Phys. Rev. Lett. 94, 248103 (2005).

    Article  CAS  Google Scholar 

  53. Touchon, M. et al. Replication-associated strand asymmetries in mammalian genomes: toward detection of replication origins. Proc. Natl. Acad. Sci. USA 102, 9836–9841 (2005).

    Article  CAS  Google Scholar 

  54. Touchon, M., Nicolay, S., Arneodo, A., d'Aubenton-Carafa, Y. & Thermes, C. Transcription-coupled TA and GC strand asymmetries in the human genome. FEBS Lett. 555, 579–582 (2003).

    Article  CAS  Google Scholar 

  55. Touchon, M., Arneodo, A., d'Aubenton-Carafa, Y. & Thermes, C. Transcription-coupled and splicing-coupled strand asymmetries in eukaryotic genomes. Nucleic Acids Res. 32, 4969–4978 (2004).

    Article  CAS  Google Scholar 

  56. Audit, B. et al. DNA replication timing data corroborate in silico human replication origin predictions. Phys. Rev. Lett. 99, 248102 (2007).

    Article  CAS  Google Scholar 

  57. Huvet, M. et al. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17, 1278–1285 (2007).

    Article  CAS  Google Scholar 

  58. Baker, A. et al. Wavelet-based method to disentangle transcription- and replication-associated strand asymmetries in mammalian genomes. Appl. Comput. Harmon. Anal. 28, 150–170 (2010).

    Article  Google Scholar 

  59. Chen, C.-L. et al. Replication-associated mutational asymmetry in the human genome. Mol. Biol. Evol. 28, 2327–2337 (2011).

    Article  CAS  Google Scholar 

  60. Arneodo, A. et al. Multi-scale coding of genomic information: from DNA sequence to genome structure and function. Phys. Rep. 498, 45–188 (2011).

    Article  CAS  Google Scholar 

  61. Audit, B. et al. Open chromatin encoded in DNA sequence is the signature of 'master' replication origins in human cells. Nucleic Acids Res. 37, 6064–6075 (2009).

    Article  CAS  Google Scholar 

  62. Baker, A. et al. Replication fork polarity gradients revealed by megabase-sized U-shaped replication timing domains in human cell lines. PLoS Comput. Biol. 8, e1002443 (2012).

    Article  CAS  Google Scholar 

  63. Phillips, J.E. & Corces, V.G. CTCF: master weaver of the genome. Cell 137, 1194–1211 (2009).

    Article  Google Scholar 

  64. Ohlsson, R., Lobanenkov, V. & Klenova, E. Does CTCF mediate between nuclear organization and gene expression? Bioessays 32, 37–50 (2010).

    Article  CAS  Google Scholar 

  65. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).

    Article  CAS  Google Scholar 

  66. Dixon, J.R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

    Article  CAS  Google Scholar 

  67. Gauthier, M.G., Norio, P. & Bechhoefer, J. Modeling inhomogeneous DNA replication kinetics. PLoS ONE 7, e32053 (2012).

    Article  CAS  Google Scholar 

  68. Mallat, S. A Wavelet Tour of Signal Processing (Academic Press, 1998).

  69. Arneodo, A., Audit, B., Decoster, N., Muzy, J.-F. & Vaillant, C. Wavelet-based multifractal formalism: application to DNA sequences, satellite images of the cloud structure and stock market data. In The Science of Disasters: Climate Disruptions, Heart Attacks, and Market Crashes (eds. Bunde, A., Kropp, J. & Schellnhuber, H.J.) 26–102 (Springer, 2002).

  70. Arneodo, A., Argoul, F., Bacry, E., Elezgaray, J. & Muzy, J.-F. Ondelettes Multifractales et Turbulences: de l'ADN aux Croissances Cristallines (Diderot éditeur, Arts et Sciences, 1995).

  71. Abry, P. Ondelettes et Turbulences (Diderot éditeur, Arts et Sciences, 1997).

  72. Arneodo, A., Bacry, E., Graves, P.V. & Muzy, J.-F. Characterizing long-range correlations in DNA sequences from wavelet analysis. Phys. Rev. Lett. 74, 3293–3296 (1995).

    Article  CAS  Google Scholar 

  73. Audit, B. et al. Long-range correlations in genomic DNA: a signature of the nucleosomal structure. Phys. Rev. Lett. 86, 2471–2474 (2001).

    Article  CAS  Google Scholar 

  74. Audit, B., Vaillant, C., Arneodo, A., d'Aubenton-Carafa, Y. & Thermes, C. Long-range correlations between DNA bending sites: relation to the structure and dynamics of nucleosomes. J. Mol. Biol. 316, 903–918 (2002).

    Article  CAS  Google Scholar 

  75. Audit, B. & Ouzounis, C.A. From genes to genomes: universal, scale-invariant properties of microbial chromosome organisation. J. Mol. Biol. 332, 617–633 (2003).

    Article  CAS  Google Scholar 

  76. Nicolay, S. et al. From scale invariance to deterministic chaos in DNA sequences: towards a deterministic description of gene organization in the human genome. Physica A 342, 270–280 (2004).

    Article  CAS  Google Scholar 

  77. Nicolay, S. et al. Low frequency rhythms in human DNA sequences: a key to the organization of gene location and orientation? Phys. Rev. Lett. 93, 108101 (2004).

    Article  CAS  Google Scholar 

  78. Nicolay, S. et al. Bifractality of human DNA strand-asymmetry profiles results from transcription. Phys. Rev. E 75, 032902 (2007).

    Article  CAS  Google Scholar 

  79. Kestener, P., Lina, J.-M., Saint-Jean, P. & Arneodo, A. Wavelet-based multifractal formalism to assist in diagnosis in digitized mammograms. Image Anal. Stereol. 20, 169–174 (2001).

    Article  Google Scholar 

  80. Caddle, L.B. et al. Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells. Chromosome Res. 15, 1061–1073 (2007).

    Article  CAS  Google Scholar 

  81. Khalil, A. et al. Chromosome territories have a highly nonspherical morphology and nonrandom positioning. Chromosome Res. 15, 899–916 (2007).

    Article  CAS  Google Scholar 

  82. Muzy, J.-F., Bacry, E. & Arneodo, A. The multifractal formalism revisited with wavelets. Int. J. Bifurc. Chaos 4, 245–302 (1994).

    Article  Google Scholar 

  83. Arneodo, A., Bacry, E. & Muzy, J.-F. The thermodynamics of fractals revisited with wavelets. Physica A 213, 232–275 (1995).

    Article  CAS  Google Scholar 

  84. Arneodo, A., Audit, B., Kestener, P. & Roux, S.G. Wavelet-based multifractal analysis. Scholarpedia 3, 4103 (2008).

    Article  Google Scholar 

  85. Conti, C. et al. Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells. Mol. Biol. Cell 18, 3059–3067 (2007).

    Article  CAS  Google Scholar 

  86. Courbet, S. et al. Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455, 557–560 (2008).

    Article  CAS  Google Scholar 

  87. Mandelbrot, B.B. The Fractal Geometry of Nature (Freeman, 1982).

Download references

Acknowledgements

We thank all the contributors to the LastWave project and in particular E. Bacry for the development of the LastWave kernel. This work was supported by the Agence National de la Recherche under projects HUGOREP (ANR PCV 2005) and REFOPOL (ANR BLANC SVSE 6), and by grants from FRM (équipe labélisée), the ARC and the Ligue contre le Cancer (Comité de Paris) to O.H.

Author information

Authors and Affiliations

Authors

Contributions

All authors contributed equally to the design and application of the protocols presented in this paper. B.A. and A.A. wrote the paper.

Corresponding author

Correspondence to Benjamin Audit.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Audit, B., Baker, A., Chen, CL. et al. Multiscale analysis of genome-wide replication timing profiles using a wavelet-based signal-processing algorithm. Nat Protoc 8, 98–110 (2013). https://doi.org/10.1038/nprot.2012.145

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.145

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing