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FAIRE-Seq (Formaldehyde-Assisted Isolation of Regulatory Elements) is a method in
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
used for determining the sequences of DNA regions in the
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...
associated with regulatory activity. The technique was developed in the laboratory of Jason D. Lieb at the
University of North Carolina The University of North Carolina is the multi-campus public university system for the state of North Carolina. Overseeing the state's 16 public universities and the NC School of Science and Mathematics, it is commonly referred to as the UNC Sys ...
, Chapel Hill. In contrast to
DNase-Seq DNase-seq (DNase I hypersensitive sites sequencing) is a method in molecular biology used to identify the location of regulatory regions, based on the genome-wide sequencing of regions sensitive to cleavage by DNase I. FAIRE-Seq is a successor of ...
, 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
chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
.


Workflow

The protocol is based on the fact that the
formaldehyde Formaldehyde ( , ) ( systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section ...
cross-linking is more efficient in
nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone, histone proteins and resembles thread wrapped around a spool. The nucleosome is the f ...
-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.


Sequencing

FAIRE-extracted DNA fragments can be analyzed in a high-throughput way using
next-generation sequencing Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation s ...
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. 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 A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in re ...
. However, this method does not allow a genome wide / high-throughput quantification of the extracted fragments.


Sensitivity

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 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.


Computational analysis

In a first step FAIRE-seq data are mapped to the reference genome of the model organism used. 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 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. 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 FAIRE-seq often brings with it. Data is typically visualized as tracks (e.g. bigWig) and can be uploaded to the UCSC genome browser. 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.


Alternative methods

There are several methods that can be used as an alternative to FAIRE-seq.
DNase-seq DNase-seq (DNase I hypersensitive sites sequencing) is a method in molecular biology used to identify the location of regulatory regions, based on the genome-wide sequencing of regions sensitive to cleavage by DNase I. FAIRE-Seq is a successor of ...
uses the ability of the DNase I enzyme to cleave free/open/accessible DNA to identify and sequence open chromatin. The subsequently developed
ATAC-seq ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique used in molecular biology to assess genome-wide chromatin accessibility. In 2013, the technique was first described as an alternative advanced method for MNase- ...
employs the Tn5 transposase, which inserts specified fragments or transposons into accessible regions of the genome to identify and sequence open chromatin.


References

{{Reflist Molecular biology techniques Gene expression