FAIRE-Seq: Difference between revisions
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== Use == |
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'''FAIRE-Seq''' (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method in [[molecular biology]] used for determining the sequences of those DNA regions in the [[genome]] associated with regulatory activity.<ref name=PMID17179217>{{cite journal|last=Giresi|first=PG|author2=Kim, J |author3=McDaniell, RM |author4=Iyer, VR |author5=Lieb, JD |title=FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.|journal=Genome Research|date=Jun 2007|volume=17|issue=6|pages=877–85|pmid=17179217|doi=10.1101/gr.5533506|pmc=1891346}}</ref> The technique was developed in the laboratory of Jason D. Lieb at the [[University of North Carolina]], Chapel Hill. In contrast to [[DNase-Seq]], the FAIRE-Seq protocol doesn't require the [[permeabilization]] of cells or isolation of nuclei, and can analyse any cell types. 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 [[chromatin]]. |
'''FAIRE-Seq''' (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method in [[molecular biology]] used for determining the sequences of those DNA regions in the [[genome]] associated with regulatory activity.<ref name=PMID17179217>{{cite journal|last=Giresi|first=PG|author2=Kim, J |author3=McDaniell, RM |author4=Iyer, VR |author5=Lieb, JD |title=FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin.|journal=Genome Research|date=Jun 2007|volume=17|issue=6|pages=877–85|pmid=17179217|doi=10.1101/gr.5533506|pmc=1891346}}</ref> The technique was developed in the laboratory of Jason D. Lieb at the [[University of North Carolina]], Chapel Hill. In contrast to [[DNase-Seq]], the FAIRE-Seq protocol doesn't require the [[permeabilization]] of cells or isolation of nuclei, and can analyse any cell types. 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 [[chromatin]]. |
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== workflow == |
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The protocol is based on the fact that the [[formaldehyde]] cross-linking is more efficient in [[nucleosome]]-bound [[DNA]] 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. |
The protocol is based on the fact that the [[formaldehyde]] cross-linking is more efficient in [[nucleosome]]-bound [[DNA]] 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. |
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=== FAIRE === |
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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 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 <ref name="PMID17179217" />. |
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=== Sequencing === |
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FAIRE-extracted DNA fragments can be analyzed in a high-throughput way using [[DNA sequencing|next-generation sequencing]] 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. |
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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.<ref>{{Cite journal |
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| last = Landt |
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| first = Stephen G. |
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| last2 = Marinov |
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| first2 = Georgi K. |
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| last3 = Kundaje |
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| first3 = Anshul |
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| last4 = Kheradpour |
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| first4 = Pouya |
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| last5 = Pauli |
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| first5 = Florencia |
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| last6 = Batzoglou |
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| first6 = Serafim |
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| last7 = Bernstein |
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| first7 = Bradley E. |
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| last8 = Bickel |
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| first8 = Peter |
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| last9 = Brown |
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| first9 = James B. |
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| date = 2012-09-01 |
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| title = ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia |
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| url = http://www.ncbi.nlm.nih.gov/pubmed/22955991 |
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| journal = Genome Research |
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| volume = 22 |
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| issue = 9 |
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| pages = 1813–1831 |
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| doi = 10.1101/gr.136184.111 |
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| issn = 1549-5469 |
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| pmc = 3431496 |
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| pmid = 22955991 |
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}}</ref><ref>{{Cite journal |
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| last = Sims |
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| first = David |
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| last2 = Sudbery |
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| first2 = Ian |
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| last3 = Ilott |
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| first3 = Nicholas E. |
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| last4 = Heger |
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| first4 = Andreas |
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| last5 = Ponting |
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| first5 = Chris P. |
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| title = Sequencing depth and coverage: key considerations in genomic analyses |
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| url = http://www.nature.com/doifinder/10.1038/nrg3642 |
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| journal = Nature Reviews Genetics |
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| volume = 15 |
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| issue = 2 |
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| pages = 121–132 |
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| doi = 10.1038/nrg3642 |
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}}</ref> In addition, a reference or input genome, which has not been cross-linked, is often sequenced alongside to determine the level of background noise. |
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Note that the extracted FAIRE-fragments can be quantified in an alternative method by using quantitative PCR (link?). However, this method does not allow a genome wide / high-throughput quantification of the extracted fragments. |
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== Sensitivity == |
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== computational analysis == |
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| ⚫ | |||
==References== |
==References== |
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Revision as of 09:13, 26 February 2016
Use
FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method in molecular biology used for determining the sequences of those DNA regions in the genome associated with regulatory activity.[1] The technique was developed in the laboratory of Jason D. Lieb at the University of North Carolina, Chapel Hill. In contrast to DNase-Seq, the FAIRE-Seq protocol doesn't require the permeabilization of cells or isolation of nuclei, and can analyse any cell types. 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 chromatin.
workflow
The protocol is based on the fact that the formaldehyde cross-linking is more efficient in nucleosome-bound DNA 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.
FAIRE
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 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 [1].
Sequencing
FAIRE-extracted DNA fragments can be analyzed in a high-throughput way using next-generation sequencing 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.
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.[2][3] In addition, a reference or input genome, which has not been cross-linked, is often sequenced alongside to determine the level of background noise.
Note that the extracted FAIRE-fragments can be quantified in an alternative method by using quantitative PCR (link?). However, this method does not allow a genome wide / high-throughput quantification of the extracted fragments.
Sensitivity
computational analysis
FAIRE-seq data are mapped to the human genome assembly and displayed as part of the ENCODE project at the UCSC Genome Browser.
References
- ^ a b Giresi, PG; Kim, J; McDaniell, RM; Iyer, VR; Lieb, JD (Jun 2007). "FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin". Genome Research. 17 (6): 877–85. doi:10.1101/gr.5533506. PMC 1891346. PMID 17179217.
- ^ 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). "ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia". Genome Research. 22 (9): 1813–1831. doi:10.1101/gr.136184.111. ISSN 1549-5469. PMC 3431496. PMID 22955991.
- ^ Sims, David; Sudbery, Ian; Ilott, Nicholas E.; Heger, Andreas; Ponting, Chris P. "Sequencing depth and coverage: key considerations in genomic analyses". Nature Reviews Genetics. 15 (2): 121–132. doi:10.1038/nrg3642.