89:
phenol-chloroform extraction. This method creates two phases, an organic and an aqueous phase. Due to their biochemical properties, the DNA fragments cross-linked to nucleosomes will preferentially sit in the organic phase. Nucleosome depleted or ‘open’ regions on the other hand will be found in the aqueous phase. By specifically extracting the aqueous phase, only nucleosome-depleted regions will be purified and enriched.
134:
the enriched signal by combining the parameter callpeak with other options like 'broad', 'broad cutoff', 'no model' or 'shift'. ZINBA is a generic algorithm for detection of enrichment in short read dataset. It thus helps in the accurate detection of signal in complex datasets having low signal-to noise ratio.
137:
BedTools is used to merge the enriched regions residing close to each other to form COREs (Cluster of open regulatory elements). This helps in the identification of chromatin accessible regions and gene regulation patterns which would have been undetectable otherwise, considering the lower resolution
133:
Next, the identification of genomic regions with open chromatin, is done by using a peak calling algorithm. Different tools offer packages to do this (e.g. ChIPOTle ZINBA and MACS2). ChIPOTle uses a sliding window of 300bp to identify statistically significant signals. In contrast, MACS2 identifies
121:
There are several aspects of FAIRE-seq that require attention when analysing and interpreting the data. For one, it has been stated that FAIRE-seq will have a higher coverage at enhancer regions over promoter regions. This is in contrast to the alternative method of DNase-seq who is known to show a
88:
FAIRE uses the biochemical properties of protein-bound DNA to separate nucleosome-depleted regions in the genome. Cells will be subjected to cross-linking, ensuring that the interaction between the nucleosomes and DNA are fixed. After sonication, the fragmented and fixed DNA is separated using a
105:
Depending on the size of the genome FAIRE-seq is performed on, a minimum of reads is required to create an appropriate coverage of the data, ensuring a proper signal can be determined. In addition, a reference or input genome, which has not been cross-linked, is often sequenced alongside to
55:, the FAIRE-Seq protocol doesn't require the permeabilization of cells or isolation of nuclei, and can analyse any cell type. In a study of seven diverse human cell types, DNase-seq and FAIRE-seq produced strong cross-validation, with each cell type having 1-2% of the human genome as open
79:
than it is in nucleosome-depleted regions of the genome. This method then segregates the non cross-linked DNA that is usually found in open chromatin, which is then sequenced. The protocol consists of cross linking, phenol extraction and sequencing the DNA in aqueous phase.
101:
techniques. In general, libraries are made by ligating specific adapters to the DNA fragments that allow them to cluster on a platform and be amplified resulting in the DNA sequences being read/determined, and this in parallel for millions of the DNA fragments.
122:
higher sensitivity towards promoter regions. In addition, FAIRE-seq has been stated to show prefers for internal introns and exons. In general it is also believed that FAIRE-seq data displays a higher background level, making it a less sensitive method.
144:
The major limitation of this method, i.e. the low signal-to-noise ratio compared to other chromatin accessibility assays, makes the computational interpretation of these data very difficult.
229:
Landt, Stephen G.; Marinov, Georgi K.; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E.; Bickel, Peter; Brown, James B. (2012-09-01).
946:
Crawford, Gregory E.; Holt, Ingeborg E.; Whittle, James; Webb, Bryn D.; Tai, Denise; Davis, Sean; Margulies, Elliott H.; Chen, YiDong; Bernat, John A. (2006-01-01).
606:
Zhang, Yong; Liu, Tao; Meyer, Clifford A.; Eeckhoute, Jérôme; Johnson, David S.; Bernstein, Bradley E.; Nusbaum, Chad; Myers, Richard M.; Brown, Myles (2008-01-01).
378:
Song, Lingyun; Zhang, Zhancheng; Grasfeder, Linda L.; Boyle, Alan P.; Giresi, Paul G.; Lee, Bum-Kyu; Sheffield, Nathan C.; Gräf, Stefan; Huss, Mikael (2011-10-01).
286:
Sims, David; Sudbery, Ian; Ilott, Nicholas E.; Heger, Andreas; Ponting, Chris P. (2014). "Sequencing depth and coverage: key considerations in genomic analyses".
889:
Boyle, Alan P.; Davis, Sean; Shulha, Hennady P.; Meltzer, Paul; Margulies, Elliott H.; Weng, Zhiping; Furey, Terrence S.; Crawford, Gregory E. (2008-01-25).
160:
employs the Tn5 transposase, which inserts specified fragments or transposons into accessible regions of the genome to identify and sequence open chromatin.
1005:"Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position"
781:
Hinrichs, A. S.; Karolchik, D.; Baertsch, R.; Barber, G. P.; Bejerano, G.; Clawson, H.; Diekhans, M.; Furey, T. S.; Harte, R. A. (2006-01-01).
549:"ZINBA integrates local covariates with DNA-seq data to identify broad and narrow regions of enrichment, even within amplified genomic regions"
156:
uses the ability of the DNase I enzyme to cleave free/open/accessible DNA to identify and sequence open chromatin. The subsequently developed
329:
Kumar, Vibhor; Muratani, Masafumi; Rayan, Nirmala Arul; Kraus, Petra; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam (2013-07-01).
1066:
182:"FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin"
110:
113:. However, this method does not allow a genome wide / high-throughput quantification of the extracted fragments.
1003:
Buenrostro, Jason D.; Giresi, Paul G.; Zaba, Lisa C.; Chang, Howard Y.; Greenleaf, William J. (2013-12-01).
48:
47:
associated with regulatory activity. The technique was developed in the laboratory of Jason D. Lieb at the
380:"Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity"
948:"Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS)"
441:
141:
Data is typically visualized as tracks (e.g. bigWig) and can be uploaded to the UCSC genome browser.
1071:
130:
In a first step FAIRE-seq data are mapped to the reference genome of the model organism used.
547:
Rashid, Naim U.; Giresi, Paul G.; Ibrahim, Joseph G.; Sun, Wei; Lieb, Jason D. (2011-01-01).
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Note that the extracted FAIRE-fragments can be quantified in an alternative method by using
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891:"High-resolution mapping and characterization of open chromatin across the genome"
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FAIRE-extracted DNA fragments can be analyzed in a high-throughput way using
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There are several methods that can be used as an alternative to FAIRE-seq.
68:
395:
246:
798:
231:"ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia"
726:"BEDTools: a flexible suite of utilities for comparing genomic features"
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Koohy, Hashem; Down, Thomas A.; Spivakov, Mikhail; Hubbard, Tim (2014).
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72:
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52:
299:
490:"ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data"
331:"Uniform, optimal signal processing of mapped deep-sequencing data"
157:
180:
Giresi, PG; Kim, J; McDaniell, RM; Iyer, VR; Lieb, JD (Jun 2007).
179:
780:
44:
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Buck, Michael J; Nobel, Andrew B; Lieb, Jason D (2005-01-01).
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1002:
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43:
used for determining the sequences of DNA regions in the
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888:
377:
328:
667:"A Comparison of Peak Callers Used for DNase-Seq Data"
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546:
285:
840:"Chromatin accessibility: a window into the genome"
437:"Chromatin accessibility: a window into the genome"
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783:"The UCSC Genome Browser Database: update 2006"
435:Tsompana, Maria; Buck, Michael J (2014-11-20).
170:
724:Quinlan, Aaron R.; Hall, Ira M. (2010-03-15).
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67:The protocol is based on the fact that the
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608:"Model-based analysis of ChIP-Seq (MACS)"
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106:determine the level of background noise.
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838:Tsompana, M; Buck, MJ (2014-11-20).
71:cross-linking is more efficient in
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138:FAIRE-seq often brings with it.
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51:, Chapel Hill. In contrast to
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1067:Molecular biology techniques
692:10.1371/journal.pone.0096303
49:University of North Carolina
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844:Epigenetics & Chromatin
442:Epigenetics & Chromatin
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907:10.1016/j.cell.2007.12.014
99:next-generation sequencing
625:10.1186/gb-2008-9-9-r137
566:10.1186/gb-2011-12-7-r67
507:10.1186/gb-2005-6-11-r97
83:
39:lements) is a method in
288:Nature Reviews Genetics
857:10.1186/1756-8935-7-33
793:(suppl 1): D590–D598.
787:Nucleic Acids Research
456:10.1186/1756-8935-7-33
126:Computational analysis
396:10.1101/gr.121541.111
247:10.1101/gr.136184.111
335:Nature Biotechnology
683:2014PLoSO...996303K
148:Alternative methods
1021:10.1038/nmeth.2688
964:10.1101/gr.4074106
799:10.1093/nar/gkj144
198:10.1101/gr.5533506
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730:Bioinformatics
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31:solation of
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23:ormaldehyde-
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618:(9): R137.
500:(11): R97.
117:Sensitivity
1061:Categories
559:(7): R67.
164:References
93:Sequencing
73:nucleosome
35:egulatory
1029:1548-7105
972:1088-9051
915:1097-4172
850:(1): 33.
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154:DNase-seq
57:chromatin
53:DNase-Seq
17:FAIRE-Seq
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825:16381938
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593:21787385
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475:25473421
422:21750106
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273:22955991
216:17179217
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63:Workflow
27:ssisted
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