## Description

The subtracks within this annotation show replication origin predictions and confidence intervals for DNA synthesis.

## Display Conventions and Configuration

This annotation follows the display conventions for composite tracks. All subtracks with checked boxes will be displayed.

## 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 and DNA was isolated from them. 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 classed as temporally specific or non-specific based on whether or not at least 50% of the accumulated signal appeared in a single time point.

The TR50 data for the temporally specific probes were then smoothed within a 95 kb window using lowess smoothing. This smoothing was done for three replicates (two biological and one technical) and for the average of the three replicates. Local minima were identified on the smoothed TR50 profiles to locate possible origins of replication. The predictions were taken from the average of the replicates and then given a width of 200 bp for display in the browser. The confidence intervals were identified as consensus sites among the three replicates with endpoints taken on the outlying replicates for each consensus site.

## 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 this track 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, D.S., Gingeras, T.R. and Dutta, A. Temporal profile of replication of human chromosomes. Proc Natl Acad Sci U S A 102(18), 6419-24 (2005).