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.
Highlights
Replication of eukaryotic genomes is an essential process that guarantees the accurate copying of genetic information before cell division
We describe the use of the LastWave open-source signal-processing command language for analyzing cellular DNA replication timing profiles
We describe the flow of signal-processing operations to obtain interactive visual analyses of DNA replication timing profiles
Summary
Replication of eukaryotic genomes is an essential process that guarantees the accurate copying of genetic information before cell division. By partitioning the genome on the basis of the apparent speed of replication (the inverse of the timing profile slope)[37], we are in the position to question the reported absence of replication origins within TTRs26,27,33 and to determine the extent of origin synchrony within CTRs. as explained in the theoretical background section, given reasonable assumptions, the described wavelet-based methodology enables us to extract fundamental parameters, such as the average replication fork polarity and the difference between the densities of replication origins and termination sites[62]. As explained in the theoretical background section, given reasonable assumptions, the described wavelet-based methodology enables us to extract fundamental parameters, such as the average replication fork polarity and the difference between the densities of replication origins and termination sites[62] In this protocol, we describe the main steps of this signal processing strategy when applied to mean replication timing profiles in HeLa cells that have been fully calibrated to real time along the S phase[37]. We are currently developing novel procedures to define, from chromosome conformation data[65], an objective segmentation of the human genome into topological chromatin domains[66], which will enable us to quantitatively assess the relationship between replication domains and chromatin domains
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