## Description

These tracks display Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) evidence as part of the four Open Chromatin track sets. 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 syntheseis of all the open chromatin assays for select cell lines can be previewed 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
  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
  Density 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 4 or less times in the genome, and uses an alignability background model to try to correct for regions where sequences cannot be aligned. For each cell type, especially important for those with an abnormal karyotype, a model to try to correct for amplifications and deletions that is based on control input data was also used.

Base Overlap Signal
  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. Also see 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 BWA (Li et al., 2010) for the NCBI 36 (hg19) assembly.

The command used for these alignments was:
  `> bwa aln -t 8 genome.fa s_1.sequence.txt.bfq > s_1.sequence.txt.sai`

Where genome.fa is the whole genome sequence and s_1.sequence.txt.bfq is one lane of sequences converted into the required bfq format.

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 4 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. Instead, a general background model was derived from the available Input data sets. This should provide corrections for sequencing biases and alignment artifacts, but will not correct for cell type specific copy number changes.

The exact command used for this step is:
  `> fseq -l 800 -v -b <bff files> -p <iff files> aligments.bed`

Where the (bff files) are the background files based on alignability, the (iff files) are the background files based on the Input experiments, and alignments.bed are a bed file of filtered sequence alignments.

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 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 containing this data can be found in the Downloads directory labeled Validation view.

## Release Notes

Release 1 (March 2011) of this track consists of a remapping of all previously released experiments to the human reference genome GRCh37/hg19 (these data were previously mapped to NCBI36/hg18; please see the Release Notes section of the hg18 Open Chromatin track for information on the NCBI36/hg18 releases of the data).

- There are 12 new FAIRE experiments in this release, on 10 new cell lines.
- New to this release is a reconfiguration of how this track is displayed in relation to other tracks from the Duke/UNC/UT-Austin/EBI group.
- A synthesis of open chromatin evidence from the three assay types was compiled for Tier 1 and 2 cell lines plus NHEK will also be added in this release and can be previewed in: Open Chromatin Synthesis.
- Enhancer and Insulator Functional assays: A subset of DNase and FAIRE regions were cloned into functional tissue culture reporter assays to test for enhancer and insulator activity. Coordinates and results from these experiments can be found here.

## Credits

These data and annotations were created by a collaboration of multiple institutions (contact: Terry Furey):

- Duke University's Institute for Genome Sciences & Policy (IGSP): Alan Boyle, Lingyun Song, Terry Furey, and Greg Crawford

- University of North Carolina at Chapel Hill: Paul Giresi and Jason Lieb

- Universty of Texas at Austin: Zheng Liu, Ryan McDaniell, Bum-Kyu Lee, and Vishy Iyer

- European Bioinformatics Insitute: Paul Flicek, Damian Keefe, and Ewan Birney

- University of Cambridge, Department of Oncology and CR-UK Cambridge Research Institute (CRI) : Stefan Graf

We thank NHGRI for ENCODE funding support.

## References

Bhinge AA, Kim J, Euskirchen GM, Snyder M, Iyer, VR. Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE). Genome Res. 2007 Jun;17(6):910-6.

Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE. High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008 Jan 25;132(2):311-22.

Boyle AP, Guinney J, Crawford GE, and Furey TS. F-Seq: a feature density estimator for high-throughput sequence tags. Bioinformatics. 2008 Nov 1;24(21):2537-8.

Buck MJ, Nobel AB, Lieb JD. ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data. Genome Biol. 2005;6(11):R97.

Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, Collins FS. DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods. 2006 Jul;3(7):503-9.

Crawford GE, Holt IE, Whittle J, Webb BD, Tai D, Davis S, Margulies EH, Chen Y, Bernat JA, Ginsburg D et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 2006 Jan;16(1):123-31.

The ENCODE Project Consortium. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007 Jun 14;447(7146):799-816.

Giresi PG, Kim J, McDaniell RM, Iyer VR, Lieb JD. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolated active regulatory elements in human chromatin. Genome Res. 2007 Jun;17(6):877-85.

Giresi PG, Lieb JD. Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). Methods. 2009 Jul;48(3):233-9.

Li H, Ruan J, and Durbin R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 2008 Nov;18(11):1851-8.

Song L and Crawfor GE. DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb. Protoc.; 2010;Issue 2.

## Data Release Policy

Data users may freely use ENCODE data, but may not, without prior consent, submit publications that use an unpublished ENCODE dataset until nine months following the release of the dataset. This date is listed in the Restricted Until column on the track configuration page and the download page. The full data release policy for ENCODE is available here.