GSE32970
GSE32970: Open Chromatin by DNaseI HS from ENCODE/OpenChrom(Duke University)
Summary:  These tracks display DNaseI hypersensitivity (HS) evidence as part of the four Open Chromatin track sets. DNaseI is an enzyme that has long been used to map general chromatin accessibility, and DNaseI "hypersensitivity" is a feature of active cis-regulatory sequences. The use of this method has led to the discovery of functional regulatory elements that include promoters, enhancers, silencers, insulators, locus control regions, and novel elements. DNaseI hypersensitivity signifies chromatin accessibility following binding of trans-acting factors in place of a canonical nucleosome. Together with FAIRE and ChIP-seq experiments, these tracks display the locations of active regulatory elements identified as open chromatin in multiple cell types (http://hgwdev.cse.ucsc.edu/cgi-bin/hgEncodeVocab?type=cellType) 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: these DNaseI HS assays and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), 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 additional verified by a second platform, high-resolution 1% ENCODE tiled microarrays supplied by NimbleGen. Overall Design: Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell).DNaseI hypersensitive sites were isolated using methods called DNase-seq or DNase-chip (Song and Crawford, 2010; Boyle et al., 2008a; Crawford et al., 2006). Briefly, cells were lysed with NP40, and intact nuclei were digested with optimal levels of DNaseI enzyme. DNaseI digested ends were captured from three different DNase concentrations, and material was sequenced using Illumina (Solexa) sequencing. DNase-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. In general, cell lines were verified if 80% of the top 50,000 peaks in one replicate are detected in the top 100,000 peaks of a second replicate. For some cell types, additional verification was performed using similar material hybridized to NimbleGen Human ENCODE tiling arrays (1% of the genome) along with the input DNA as reference (DNase-chip). A more detailed protocol is available at http://hgwdev.cse.ucsc.edu/ENCODE/protocols/general/Duke_DNase_protocol.pdf.The read length for sequences from DNase-seq are 20 bases long due to a MmeI cutting step of the approximately >50kb DNA fragments extracted after DNaseI digestion. 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 convert 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 at http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeOpenChromDnase/supplemental/). The mappings of these short reads to the genome are available for download at http://hgwdev.cse.ucsc.edu/cgi-bin/hgFileUi?g=wgEncodeOpenChromDnase.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 600 -v -f 0 -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 DNaseI HS sites (peaks) were identified from DNase-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 containing this data can be found in the Downloads directory (http://hgwdev.cse.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeOpenChromDnase) labeled Validation view.Release 1 (April 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 (http://hgwdev.cse.ucsc.edu/cgi-bin/hgTrackUi?db=hg18&g=wgEncodeChromatinMap) for information on the NCBI36/hg18 releases of the data). There are 21 new DNaseI experiments in this release, on 19 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 (http://genome-preview.cse.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeOpenChromSynth). 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 at http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeOpenChromDnase/supplemental/.