Description

These tracks display Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) evidence as part of the four Open Chromatin track sets (see below). FAIRE is a method to isolate and identify nucleosome-depleted regions of the genome. FAIRE was initially discovered in yeast and subsequently shown to identify active regulatory elements in human cells (Giresi et al., 2007). Similar to DNaseI HS, FAIRE appears to identify functional regulatory elements that include promoters, enhancers, silencers, insulators, locus control regions and novel elements.

Together with DNaseI HS and ChIP-seq experiments, these tracks display the locations of active regulatory elements identified as open chromatin in multiple cell types from the Duke, UNC-Chapel Hill, UT-Austin, and EBI ENCODE group. Within this project, open chromatin was identified using two independent and complementary methods: DNaseI hypersensitivity (HS) and these FAIRE assays, combined with chromatin immunoprecipitation (ChIP) for select regulatory factors. DNaseI HS and FAIRE provide assay cross-validation with commonly identified regions delineating the highest confidence areas of open chromatin. ChIP assays provide functional validation and preliminary annotation of a subset of open chromatin sites. Each method employed Illumina (formerly Solexa) sequencing by synthesis as the detection platform. The Tier 1 and Tier 2 cell types were additionally verified by a second platform, high-resolution 1% ENCODE tiled microarrays supplied by NimbleGen.

Other Open Chromatin track sets:

• Data for the DNase experiments can be found in Duke DNaseI HS.
• Data for the ChIP experiments can be found in UTA TFBS.
• A synthesis of all the open chromatin assays for select cell lines can be found in Open Chrom Synth.

Display Conventions and Configuration

This track is a multi-view composite track that contains a single data type with multiple levels of annotation (views). For each view, there are multiple subtracks representing different cell types that display individually on the browser. Instructions for configuring multi-view tracks are here. Chromatin data displayed here represents a continuum of signal intensities. The Leib lab recommends setting the "Data view scaling: auto-scale" option when viewing signal data in full mode to see the full dynamic range of the data. Note that in regions that do not have open chromatin sites, autoscale will rescale the data and inflate the background signal, making the regions appear noisy. Changing back to fixed scale will alleviate this issue. In general, for each experiment in each of the cell types, the UNC FAIRE tracks contain the following views:

Peaks

Peaks are regions of enriched signal in FAIRE experiments. Peaks were called based on signals created using F-Seq, a software program developed at Duke (Boyle et al., 2008b). Significant regions were determined by fitting the data to a gamma distribution to calculate p-values. Contiguous regions where p-values were below a 0.05/0.01 threshold were considered significant. The solid vertical line in the peak represents the point with highest signal.

F-Seq Density Signal

F-Seq Density Signal is a graph (wiggle) of signal enrichment calculated using F-Seq for the combined set of sequences from all replicates. F-Seq employs Parzen kernel density estimation to create base pair scores (Boyle et al., 2008b). This method does not look at fixed-length windows, but rather weights contributions of nearby sequences in proportion to their distance from that base. It only considers sequences aligned four or less times in the genome, and uses an alignability background model to try to correct for regions where sequences cannot be aligned. A model based on control input data was also used for each cell type to try to correct for amplifications and deletions, especially important for cells with an abnormal karyotype.

Base Overlap Signal

Base Overlap Signal is an alternative version of the F-Seq Density Signal track annotation that provides a higher resolution view of the raw sequence data. This track also includes the combined set of sequences from all replicates. For each sequence, the aligned read is extended 5 bp in both directions from its 5' aligned end where DNase cut the DNA. The score at each base pair represents the number of extended fragments that overlap the base pair.

Tracks displayed in this track are the results of pooled replicates. The raw sequence and alignment files for each replicate are available for download.

Metadata for a particular subtrack can be found by clicking the down arrow in the list of subtracks.

Methods

Cells were grown according to the approved ENCODE cell culture protocols.

FAIRE was performed (Giresi et al., 2007) by cross-linking proteins to DNA using 1% formaldehyde solution, and the complex was sheared using sonication. Phenol/chloroform extractions were performed to remove DNA fragments cross-linked to protein. The DNA recovered in the aqueous phase was sequenced using an Illumina (Solexa) sequencing system. FAIRE-seq data for Tier 1 and Tier 2 cell lines were verified by comparing multiple independent growths (replicates) and determining the reproducibility of the data. For some cell types additional verification was performed using the same material, but hybridized to NimbleGen Human ENCODE tiling arrays (1% of the genome) along with the input DNA as reference (FAIRE-chip). A more detailed protocol is available here and in the references below (Giresi et al., 2009).

DNA fragments isolated by FAIRE are 100-200 bp in length, with the average length being 134 bp. Sequences from each experiment were aligned to the genome using Burrows-Wheeler Aligner (BWA) (Li et al., 2010) for the NCBI 36 (hg19) assembly.

Sequences from multiple lanes are combined for a single replicate using the bwa samse command, and converted in the sam/bam format using SAMtools.

Only those that aligned to four or fewer locations were retained. Other sequences were also filtered based on their alignment to problematic regions (such as satellites and rRNA genes - see supplemental materials). The mappings of these short reads to the genome are available for download.

The resulting digital signal was converted to a continuous wiggle track using F-Seq that employs Parzen kernel density estimation to create base pair scores (Boyle et al., 2008b). Input data has been generated for several cell lines. These are used directly to create a control/background model used for F-Seq when generating signal annotations for these cell lines. These models are meant to correct for sequencing biases, alignment artifacts, and copy number changes in these cell lines. Input data is not being generated directly for other cell lines. For cell lines for which there is no input experiment available, the peaks were generated using the control of generic_male or generic_female, as an attempt to create a general background based on input data from several cell types. These files are in "iff" format, which is used when calling peaks with F-seq software, and can be downloaded from the production lab directly from under the section titled "Copy number / karyotype correction." Using a general background model derived from the available Input data sets provided corrections for sequencing biases and alignment artifacts, but will not correct for cell-type-specific copy number changes.

Discrete FAIRE sites (peaks) were identified from FAIRE-seq F-seq density signal. Significant regions were determined by fitting the data to a gamma distribution to calculate p-values. Contiguous regions where p-values were below a 0.05/0.01 threshold were considered significant.

Data from the high-resolution 1% ENCODE tiled microarrays supplied by NimbleGen were normalized using the Tukey biweight normalization, and peaks were called using ChIPOTle (Buck et al., 2005) at multiple levels of significance. Regions matched on size to these peaks that were devoid of any significant signal were also created as a null model. These data were used for additional verification of Tier 1 and Tier 2 cell lines by ROC analysis. Files labeled Validation view containing this data are available for download.

Release Notes

Release 2 (September 2012) of this track consists of 12 new experiments, including 11 new cell lines.