This track shows a tiling path of PCR amplicons, along with their raw DNaseI sensitivity scores, across all ENCODE regions. It is one of a set of tracks that annotate continuous DNaseI sensitivity measurements and DNaseI hypersensitive sites (HSs) over the ENCODE regions. DNaseI has long been used to map general chromatin accessibility and the DNaseI "hyperaccessibility" or "hypersensitivity" that is a universal feature of active cis-regulatory sequences. The data were produced using quantitative chromatin profiling (QCP) (Dorschner et al., 2004).
DNaseI-treated and untreated chromatin samples from the following cell lines/phenotypes were studied:
The display is separated into "odd" and "even" amplicons, to provide a visually distinct appearance among amplicons, so that adjacent amplicons are always in different subtracks. The details page for each amplicon reveals its start/stop coordinates and its raw DNaseI sensitivity score. The score is calculated by the formula (copies in DNaseI-treated / copies in DNaseI-untreated) * 1000.
The graphical display may be filtered to show only those items with unnormalized scores that meet or exceed a certain threshhold. To set a threshhold, type the minimum score into the text box at the top of the description page.
QCP was performed as described in Dorschner et al. PCR amplicons of ~250 bp in size were tiled end-to-end across the study regions. An amplicon tiling path has been computed over all regions and is available through UniSTS.
Chromatin preparation and DNaseI treatment were performed on the cell types list above as described in Dorschner et al. High-throughput real-time PCR was used to quantify DNaseI at each amplicon by measuring copies remaining in DNaseI-treated vs. untreated samples. The results were then analyzed with a statistical algorithm to compute the moving baseline of mean DNaseI sensitivity and to identify outliers that correspond with DNaseI hypersensitive sites.
QCP measurements were performed in replicate (6X) on pooled biological replicate samples. Validation of the results was carried out by conventional DNaseI hypersensitivity assays using end-labeling/Southern blotting. A total of 1.17 Mb have been evaluated by conventional assay.
The specificity was defined as the number of true negative evaluable QCP amplicons divided by the sum of the true negatives plus false positives. Using 246.2 Kb from ENm002, the specificity was calculated to be 0.997. The sensitivity of the QCP assay was calculated as the true positives divided by the sum of the true positives plus false negatives. The sensitivity measured for ENm002 was 0.9487.
Data generation, analysis, and validation were performed jointly by groups at Regulome Corporation and the University of Washington (UW) in Seattle.
Regulome Corp.: Michael O. Dorschner, Richard Humbert, Peter J. Sabo, Anthony Shafer, Jeff Goldy, Molly Weaver, Kristin Lee, Fidencio Neri, Brendan Henry, Mike Hawrylycz, Paul Tittel, Jim Wallace, Josh Mack, Janelle Kawamoto, John A. Stamatoyannopoulos.
UW Medical Genetics: Patrick Navas, Man Yu, Hua Cao, Brent Johnson, Ericka Johnson, George Stamatoyannopoulos.
UW Genome Sciences: Scott Kuehn, Robert Thurman, William S. Noble.
Dorschner, M.O., Hawrylycz, M., Humbert, R., Wallace, J.C., Shafer, A., Kawamoto, J., Mack, J., Hall, R., Goldy, J., Sabo, P.J. et al. High-throughput localization of functional elements by quantitative chromatin profiling. Nat Methods 1(3), 219-25 (2004).