Note: ENCODE Project
Description
These tracks display Formaldehyde-Assisted Isolation of Regulatory Elements
(FAIRE) evidence produced as part of the ENCODE Project Consortium (ENCODE
Project Consortium, 2012).
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.
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 GRCh37 (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 was generated for several
cell lines. These were used directly to create a control/background model used
for F-Seq when generating signal annotations for these cell lines.
These models were meant to correct for sequencing biases, alignment artifacts,
and copy number changes in these cell lines. Input data was not generated
directly for other cell lines. Instead, a general background model was derived
from the available Input data sets. This provided corrections for
sequencing biases and alignment artifacts, but did not correct for cell
type specific copy number changes.
- The exact command used for this step was:
> 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 the 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 0.1 threshold were considered significant.
Uniform signal was generated by processing the aligned reads using the align2rawsignal "Wiggler" software (see http://code.google.com/p/align2rawsignal for details and settings). The method accounts for the depth of sequencing, the mappability of the genome (based on read length and ambiguous bases) and different fragment length shifts for the different datasets being combined. It also differentiates between positions that showed zero signal simply because they are unmappable and positions that are mappable but have no reads.
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.
Credits
These data and annotations were created by a collaboration of multiple
institutions (contact:
Terry Furey):
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.
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.
ENCODE Project Consortium.
An integrated encyclopedia of DNA elements in the human genome.
Nature 2012 Sep 6;489(7414):57-74.
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 Crawford 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.
There is no restriction on the use of these specific tracks.
Contact
Terry Furey
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