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.
Similar content being viewed by others
References
Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).
DePamphilis, M.L. (ed). DNA Replication and Human Disease (Cold Spring Harbor Laboratory Press, 2006).
Aladjem, M.I. Replication in context: dynamic regulation of DNA replication patterns in metazoans. Nat. Rev. Genet. 8, 588–600 (2007).
Hamlin, J.L., Mesner, L.D. & Dijkwel, P.A. A winding road to origin discovery. Chromosome Res. 18, 45–61 (2010).
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).
Lucas, I. et al. High-throughput map** of origins of replication in human cells. EMBO Rep. 8, 770–777 (2007).
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).
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).
Karnani, N., Taylor, C.M. & Dutta, A. Microarray analysis of DNA replication timing. Methods Mol. Biol. 556, 191–203 (2009).
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).
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).
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).
Hyrien, O., Maric, C. & Méchali, M. Transition in specification of embryonic metazoan DNA replication origins. Science 270, 994–997 (1995).
Gerbi, S.A. & Bielinsky, A.K. DNA replication and chromatin. Curr. Opin. Genet. Dev. 12, 243–248 (2002).
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).
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).
Fisher, D. & Méchali, M. Vertebrate HoxB gene expression requires DNA replication. EMBO J. 22, 3737–3748 (2003).
Gilbert, D.M. Making sense of eukaryotic DNA replication origins. Science 294, 96–100 (2001).
MacAlpine, D.M. & Bell, S.P. A genomic view of eukaryotic DNA replication. Chromosome Res. 13, 309–326 (2005).
Méchali, M. Eukaryotic DNA replication origins: many choices for appropriate answers. Nat. Rev. Mol. Cell Biol. 11, 728–738 (2010).
Gilbert, D.M. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat. Rev. Genet. 11, 673–684 (2010).
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).
Raghuraman, M.K. et al. Replication dynamics of the yeast genome. Science 294, 115–121 (2001).
MacAlpine, D.M., Rodriguez, H.K. & Bell, S.P. Coordination of replication and transcription along a Drosophila chromosome. Genes Dev. 18, 3094–3105 (2004).
Eaton, M.L. et al. Chromatin signatures of the Drosophila replication program. Genome Res. 21, 164–174 (2011).
Farkash-Amar, S. et al. Global organization of replication time zones of the mouse genome. Genome Res. 18, 1562–1570 (2008).
Hiratani, I. et al. Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6, e245 (2008).
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).
Woodfine, K. et al. Replication timing of the human genome. Hum. Mol. Genet. 13, 191–202 (2004).
Jeon, Y. et al. Temporal profile of replication of human chromosomes. Proc. Natl. Acad. Sci. USA 102, 6419–6424 (2005).
Woodfine, K. et al. Replication timing of human chromosome 6. Cell Cycle 4, 172–176 (2005).
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).
Desprat, R. et al. Predictable dynamic program of timing of DNA replication in human cells. Genome Res. 19, 2288–2299 (2009).
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).
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).
Weddington, N. et al. Replicationdomain: a visualization tool and comparative database for genome-wide replication timing data. BMC Bioinformatics 9, 530 (2008).
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).
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).
Yaffe, E. et al. Comparative analysis of DNA replication timing reveals conserved large-scale chromosomal architecture. PLoS Genet. 6, e1001011 (2010).
Friedman, K.L., Brewer, B.J. & Fangman, W.L. Replication profile of Saccharomyces cerevisiae chromosome VI. Genes Cells 2, 667–678 (1997).
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).
Rhind, N. DNA replication timing: random thoughts about origin firing. Nat. Cell Biol. 8, 1313–1316 (2006).
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).
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).
Rhind, N., Yang, S.C. & Bechhoefer, J. Reconciling stochastic origin firing with defined replication timing. Chromosome Res. 18, 35–43 (2010).
Retkute, R., Nieduszynski, C.A. & de Moura, A. Dynamics of DNA replication in yeast. Phys. Rev. Lett. 107, 068103 (2011).
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).
Hyrien, O. & Goldar, A. Mathematical modelling of eukaryotic DNA replication. Chromosome Res. 18, 147–161 (2010).
Goldar, A., Marsolier-Kergoat, M.-C. & Hyrien, O. Universal temporal profile of replication origin activation in eukaryotes. PLoS ONE 4, e5899 (2009).
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).
Goldar, A., Labit, H., Marheineke, K. & Hyrien, O. A dynamic stochastic model for DNA replication initiation in early embryos. PLoS ONE 3, e2919 (2008).
Brodie of Brodie, E.-B. et al. From DNA sequence analysis to modeling replication in the human genome. Phys. Rev. Lett. 94, 248103 (2005).
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).
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).
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).
Audit, B. et al. DNA replication timing data corroborate in silico human replication origin predictions. Phys. Rev. Lett. 99, 248102 (2007).
Huvet, M. et al. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17, 1278–1285 (2007).
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).
Chen, C.-L. et al. Replication-associated mutational asymmetry in the human genome. Mol. Biol. Evol. 28, 2327–2337 (2011).
Arneodo, A. et al. Multi-scale coding of genomic information: from DNA sequence to genome structure and function. Phys. Rep. 498, 45–188 (2011).
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).
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).
Phillips, J.E. & Corces, V.G. CTCF: master weaver of the genome. Cell 137, 1194–1211 (2009).
Ohlsson, R., Lobanenkov, V. & Klenova, E. Does CTCF mediate between nuclear organization and gene expression? Bioessays 32, 37–50 (2010).
Lieberman-Aiden, E. et al. Comprehensive map** of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).
Dixon, J.R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).
Gauthier, M.G., Norio, P. & Bechhoefer, J. Modeling inhomogeneous DNA replication kinetics. PLoS ONE 7, e32053 (2012).
Mallat, S. A Wavelet Tour of Signal Processing (Academic Press, 1998).
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).
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).
Abry, P. Ondelettes et Turbulences (Diderot éditeur, Arts et Sciences, 1997).
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).
Audit, B. et al. Long-range correlations in genomic DNA: a signature of the nucleosomal structure. Phys. Rev. Lett. 86, 2471–2474 (2001).
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).
Audit, B. & Ouzounis, C.A. From genes to genomes: universal, scale-invariant properties of microbial chromosome organisation. J. Mol. Biol. 332, 617–633 (2003).
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).
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).
Nicolay, S. et al. Bifractality of human DNA strand-asymmetry profiles results from transcription. Phys. Rev. E 75, 032902 (2007).
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).
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).
Khalil, A. et al. Chromosome territories have a highly nonspherical morphology and nonrandom positioning. Chromosome Res. 15, 899–916 (2007).
Muzy, J.-F., Bacry, E. & Arneodo, A. The multifractal formalism revisited with wavelets. Int. J. Bifurc. Chaos 4, 245–302 (1994).
Arneodo, A., Bacry, E. & Muzy, J.-F. The thermodynamics of fractals revisited with wavelets. Physica A 213, 232–275 (1995).
Arneodo, A., Audit, B., Kestener, P. & Roux, S.G. Wavelet-based multifractal analysis. Scholarpedia 3, 4103 (2008).
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).
Courbet, S. et al. Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455, 557–560 (2008).
Mandelbrot, B.B. The Fractal Geometry of Nature (Freeman, 1982).
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
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
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights 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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2012.145
- Springer Nature Limited
This article is cited by
-
Genome-wide map** of individual replication fork velocities using nanopore sequencing
Nature Communications (2022)
-
FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing
Genome Biology (2020)
-
Multi-scale structural community organisation of the human genome
BMC Bioinformatics (2017)
-
A journey through the microscopic ages of DNA replication
Protoplasma (2017)
-
Replication landscape of the human genome
Nature Communications (2016)