ATAC-seq
ATAC-seq stands for Assay for Transposase-Accessible Chromatin with high throughput sequencing. It is a technique used in molecular biology to study chromatin accessibility. The technique was first described in 2013,[1] as an alternative or complementary method to MNase-seq (sequencing of micrococcal nuclease sensitive sites), FAIRE-seq and DNAse-seq. It aims to identify accessible DNA regions, equivalent to DNase I hypersensitive sites.
Description
The key part of the ATAC-seq procedure is the action of the transposase Tn5 on the genomic DNA of the sample.[2] Transposases are enzymes catalyzing the movement of transposons to other parts in the genome. While naturally occurring transposases have a low level of activity, ATAC-seq employs a mutated hyperactive transposase. The high activity allows for highly efficient cutting of exposed DNA and simultaneous ligation of specific sequences, called adapters. Adapter-ligated DNA fragments are then isolated, amplified by PCR and used for next generation sequencing [1](see external reference for explanatory image).
Usage and computational analysis
Transposons are believed to incorporate preferentially into genomic regions free of nucleosomes (nucleosome-free regions) or stretches of exposed DNA in general.[1] Thus enrichment of sequences from certain loci in the genome indicates absence of DNA-binding proteins or nucleosome in the region.
An ATAC-seq experiment will typically produce millions of next generation sequencing reads that can be successfully mapped on the reference genome. Each sequencing read points to a position on the genome where one transposition (or cutting) event took place during the experiment. One can then assign a cut count for each genomic position and create a signal with base-pair resolution.
Regions of the genome where DNA was accessible during the experiment will contain significantly more sequencing reads (since that is where the transposase preferentially acts), and form peaks in the ATAC-seq signal that are detectable with peak calling tools. These regions can be further categorized into the various regulatory element types - promoters, enhancers, insulators , etc- by taking into account further information , like their distance from a Transcription Start Site or data from other experiments.[1] Inside the regions where the ATAC-seq signal is enriched, one can also observe sub-regions with depleted signal. These subregions, typically only a few base pairs long, are considered to be “footprints” of DNA-binding proteins. These proteins will protect the DNA strand from transposase cleavage and will consequently cause a depletion in the signal.
An ATACseq experiment can also be used to infer nucleosome positions.[3]
Advantages
The advantages of ATAC-seq include:
- Low requirements on the amount of the biological sample. 50,000 cells are sufficient for this technique, as opposed to others like MNase-seq or DNAse-seq that require at least 1,000-fold more material.[2]
- Speed: The whole protocol requires 3 hours in total.[2]
References
- ^ a b c d Buenrostro, Jason D; Giresi, Paul G; Zaba, Lisa C; Chang, Howard Y; Greenleaf, William J (6 October 2013). "Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position". Nature Methods. 10 (12): 1213–1218. doi:10.1038/nmeth.2688.
- ^ a b c Buenrostro, Jason D.; Wu, Beijing; Chang, Howard Y.; Greenleaf, William J. (January 2015). "ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide". Current Protocols in Molecular Biology. doi:10.1002/0471142727.mb2129s109.
- ^ Schep, Alicia N.; Buenrostro, Jason D.; Denny, Sarah K.; Schwartz, Katja; Sherlock, Gavin; Greenleaf, William J. (2015-08-27). "Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions". Genome Research: gr.192294.115. doi:10.1101/gr.192294.115. ISSN 1088-9051. PMC 4617971. PMID 26314830.