#Okazaki fragment mapping verification#
#Okazaki fragment mapping windows#

The bottom panel shows the location of initiation zones (IZ) as determined by Okazaki fragment sequencing (OK-seq) (25).

#Okazaki fragment mapping windows#

The middle section shows replication timing profiles in HeLa cells taken from (46) and visualised on IGV, representing the genome replication timing windows of six consecutive stages of S phase progression, from S1 (earliest) to S6 (latest). the intersects at E = 1 × 10e-5, as determined in Figure 2). Replication origins activated in vitro were called by SICER without input green bars show peaks that are only present in both IP libraries (i.e. ( A) Positions of sequencing reads are visualised on the integrated genome viewer (IGV) for the two independent biological replicates of immunoprecipitated nascent DNA (IP-A and IP-B). Overall counts of peaks are indicated.Īctivated origins are located predominantly in early-replicating domains of the human genome. ( C) Venn diagrams of overlap between enrichment peaks called by two modes of SICER in the human genome for libraries A and B (IP A and IP B). Positions of reference genes (blue), transcription directionalities (black arrows), and primer sites used for qPCR analyses (black, origin sites grey, background sites) are indicated. the intersect) are shown for both peak-calling strategies as indicated. The areas of peaks present in both IP-A and IP-B (i.e. Peaks of nascent DNA enrichment were called by SICER, either by integrating the input DNA (w/i blue bars), or not doing so (green bars). Positions of sequencing reads are visualised on the integrated genome viewer (IGV) for total input control DNA (input) and two independent biological replicates of immunoprecipitated nascent DNA (IP-A and IP-B).

#Okazaki fragment mapping verification#

( B) Verification of origin positions by initiation site sequencing (ini-seq). Sequences for the PCR primer pairs are in Supplementary Table S1, and their genomic locations are mapped in panel B. Data are expressed as proportions of 0.1% amount of input DNA, mean values of triplicate determinations are shown for one IP experiment. Immunoprecipitated DNA was quantified by real-time PCR, using primer pairs located at the replication origins located near the promoters of the MYC, MCM4 and TOP1 gene loci (origin), and at corresponding non-origin background sites (bg). ( A) Validation of immunoprecipitation by quantification of nascent DNA at established replication origins. Published by Oxford University Press on behalf of Nucleic Acids Research.ĭetermination of replication origin locations by initiation site sequencing (ini-seq). Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq) (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins.