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Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing
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Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing
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Journal Article
Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing
2023
Overview
Studying the dynamics of genome replication in mammalian cells has been historically challenging. To reveal the location of replication initiation and termination in the human genome, we developed Okazaki fragment sequencing (OK-seq), a quantitative approach based on the isolation and strand-specific sequencing of Okazaki fragments, the lagging strand replication intermediates. OK-seq quantitates the proportion of leftward- and rightward-oriented forks at every genomic locus and reveals the location and efficiency of replication initiation and termination events. Here we provide the detailed experimental procedures for performing OK-seq in unperturbed cultured human cells and budding yeast and the bioinformatics pipelines for data processing and computation of replication fork directionality. Furthermore, we present the analytical approach based on a hidden Markov model, which allows automated detection of ascending, descending and flat replication fork directionality segments revealing the zones of replication initiation, termination and unidirectional fork movement across the entire genome. These tools are essential for the accurate interpretation of human and yeast replication programs. The experiments and the data processing can be accomplished within six days. Besides revealing the genome replication program in fine detail, OK-seq has been instrumental in numerous studies unravelling mechanisms of genome stability, epigenome maintenance and genome evolution.
A protocol for quantitative genome-wide detection of replication initiation, fork progression and termination, based on purification and strand-specific sequencing of Okazaki fragments isolated from mammalian or yeast cells.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
