## Description

The subtracks within this annotation show replication origins identified using the nascent strand method (Ori-NS), the bubble trapping method (Ori-Bubble) and the TR50 local minima method (Ori-TR50). Tracks are available for HeLa cells (cervix carcinoma) for all methods and GM06990 cells (lymphoblastoid) for Ori-NS.

## Display Conventions and Configuration

This annotation follows the display conventions for composite tracks. To show only selected subtracks within this annotation, uncheck the boxes next to the tracks you wish to hide.

## Nascent Strand Method (Ori-NS)

### Description

ENCODE region-wide mapping of replication origins was performed. Origin-centered nascent-strands purified from HeLa and GM06990 cell lines were hybridized to Affymetrix ENCODE tiling arrays.

### Methods

Cells in their exponential stage of growth were labeled, in culture, with bromodeoxyuridine (BrdU) for 30 mins. DNA was then isolated from the cells. Nascent strands of 0.5-2.5 kb synthesized with incorporation of BrdU, representing the replication origins, were purified using a sucrose gradient followed by immunoprecipitation with BrdU antibody (Giacca et al., 1997). The purified nascent strands were amplified and then hybridized to Affymetrix ENCODE tiling arrays, which have 25-mer probes tiled every 22 bp, on average, in the non-repetitive sequence of the ENCODE regions. As an experimental control, genomic DNA was hybridized to arrays independently.

Replication origins were identified by estimating the significance of the enrichment of nascent strands DNA (treatment) signal over genomic DNA (control) signal in a sliding window of 1000 bp. An estimate of significance in the window was calculated by computing the p-value using the Wilcoxon Rank-Sum test over all three biological replicates and control signal estimates in that window. The origins (Ori-NS) represented in the subtrack are the genomic regions that showed a signal enrichment pValue <= 0.001.

### Verification

The origin mapping experiments were completed for three biological sets.

### Credits

Data generation and analysis for the subtracks using the Ori-NS method were performed by the DNA replication group in the Dutta Lab at the University of Virginia: Neerja Karnani, Christopher Taylor, Ankit Malhotra, Gabe Robins and Anindya Dutta.

Christopher Taylor and Neerja Karnani prepared the data for presentation in the UCSC Genome Browser.

### References

Giacca M, Pelizon C, Falaschi A. Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction. Methods. 1997;13(3):301-12.

## Bubble Trapping Method (Ori-Bubble)

### Description

ENCODE region-wide mapping of replication origins in HeLa cells was performed by the bubble trapping method. Replication origins were identified by hybridization to Affymetrix ENCODE tiling arrays.

### Methods

The bubble trapping method works on the principle that circular plasmids can be trapped in gelling agarose followed by the application of electrical current for a prolonged period of time (see Mesner et al. 2006 for more details). Entrapment occurs by an apparent physical linkage of the circular DNA with the agarose matrix. The circular bubble component of the DNA replication intermediates was therefore enriched by agarose trapping. After recovery from the agarose gel, a library of the entrapped DNA was formed by DNA cloning. Subsequently, DNA from the library was labeled and hybridized to Affymetrix ENCODE tiling arrays, which have 25-mer probes tiled every 22 bp on average in the non-repetitive ENCODE regions. As an experimental control, genomic DNA was hybridized to arrays independently.

Replication origins were identified by estimating the significance of the enrichment of the bubble-trapped DNA (treatment) signal over genomic DNA (control) signal in a sliding window of 10,000 bp. An estimate of significance in the window was calculated by computing the p-value using the Wilcoxon Rank-Sum test over all three biological replicates and the control signal estimates in that window. The origins (Ori-Bubble) hence represented in the UCSC browser track are the genomic regions that showed a signal enrichment pValue <= 0.001.

### Verification

The origin mapping experiments were completed for two biological sets.

### Credits

Data generation and analysis for the subtrack using the Ori-bubble method were performed by the DNA replication group in the Dutta Lab and Hamlin Lab at the University of Virginia: Neerja Karnani, Larry Mesner, Christopher Taylor, Ankit Malhotra, Gabe Robins, Anindya Dutta and Joyce Hamlin.

Neerja Karnani and Christopher Taylor prepared the data for presentation in the UCSC Genome Browser.

### References

Mesner LD, Crawford EL, Hamlin JL. Isolating apparently pure libraries of replication origins from complex genomes. Mol Cell. 2006 Mar 3;21(5):719-26.

## TR50 local minima method (Ori-TR50)

### Description

ENCODE region-wide mapping of replication origins in HeLa cells was performed by the TR50 local minima method. Replication origins were identified by hybridization to Affymetrix ENCODE tiling arrays.

### Methods

The experimental strategy adopted to map this profile involved isolation of replication products from HeLa cells synchronized at the G1-S boundary by thymidine-aphidicolin double block. Cells released from the block were labeled with BrdU at every two-hour interval of the 10 hours of S-phase. Subsequently, DNA was isolated from the cells. The heavy-light (H/L) DNA representing the pool of DNA replicated during each two-hour labeling period was separated from the unlabeled DNA by double cesium chloride density gradient centrifugation. The purified H/L DNA was then hybridized to a high-density genome-tiling Affymetrix array comprised of all unique probes within the ENCODE regions.

The time of replication of 50% (TR50) of each microarray probe was calculated by accumulating the sum over the five time points and linearly interpolating the time when 50% was reached. Each probe was also classified as showing temporally specific replication (all alleles replicating together within a two-hour window) or temporally non-specific replication (at least one allele replicating apart from the others by at least a two hour difference).

The TR50 data for the temporally specific probes was then smoothed within a 60 kb window using lowess smoothing. Local minima (within a 30 kb window) on the smoothed TR50 curve were identified which had at least 30 probes in the window on both sides of the minimum to locate possible origins of replication. A confidence value was calculated for each site as the average difference from the value of the local minimum of all TR50 values falling into the 30 kb window.

### Verification

The replication experiments were completed for two biological sets and a technical replicate in the HeLa adherent cell line.

### Credits

Data generation and analysis for the subtrack using the Ori-TR50 method were performed by the DNA replication group in the Dutta Lab at the University of Virginia: Neerja Karnani, Christopher Taylor, Hakkyun Kim, Louis Lim, Ankit Malhotra, Gabe Robins and Anindya Dutta.

Neerja Karnani and Christopher Taylor prepared the data for presentation in the UCSC Genome Browser.

### References

Jeon Y, Bekiranov S, Karnani N, Kapranov P, Ghosh S, MacAlpine D, Lee C, Hwang DS, Gingeras TR, Dutta A. Temporal profile of replication of human chromosomes. Proc Natl Acad Sci U S A. 2005 May 3;102(18):6419-24.