| 词条 | ATAC-seq |
| 释义 |
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique used in molecular biology to assess genome-wide chromatin accessibility(1). In 2013, the technique was first described as an alternative advanced method for MNase-seq (sequencing of micrococcal nuclease sensitive sites), FAIRE-seq and DNAse-seq (1). ATAC-seq is an emerging technique that’s gaining popularity among researchers from diverse backgrounds as it aids in a fast and sensitive analysis of the epigenome compared to DNase-seq or MNase-seq (2,3,4). The applications of ATAC-seq in enhancing the functional genomics field have been explored in recent literature in hopes to understand epigenetic regulation in the context of disease development and cell differentiation. Indeed, ATAC-seq is becoming an essential tool in epigenetics and genome-regulation research and a standard part of epigenetic analysis. It has been successfully adapted to efficiently identify open chromatin and identify regulatory elements across the genome. DescriptionATAC-seq identifies accessible DNA regions by probing open chromatin with hyperactive mutant Tn5 transposase that inserts sequencing adapters into open regions of the genome. The mutant Tn5 transposase excises any sufficiently long DNA in a process called tagmentation: the simultaneous fragmentation and tagging of DNA performed by Tn5 transposase pre-loaded with sequencing adaptors. The tagged DNA fragments are then purified, amplified by PCR and sent for sequencing. Sequencing reads can then be used to infer regions of increased accessibility as well as to map regions of transcription-factor binding sites and nucleosome positions. The key element of the ATAC-seq procedure is the action of the transposase Tn5 on the genomic DNA of the sample. Transposases are enzymes catalyzing the movement of transposons to other parts in the genome (5). While naturally occurring transposases have a low level of activity, ATAC-seq employs a mutated hyperactive transposase(5). ApplicationsThe most common use of ATAC-Seq is in nucleosome mapping experiments (3). To this end, ATAC-Seq analysis has been used to investigate a number of chromatin-related signatures such as the genome-wide chromatin accessibility landscape in human cancer (6) and enhancer prediction. The utility of high-resolution enhancer mapping ranges from studying the evolutionary divergence of enhancer usage (e.g. between chimps and humans) during development (7) and uncovering a lineage-specific enhancer map used during blood cell differentiation (8). Most recently, ATAC-Seq has been used to reveal a genome-wide decrease in chromatin accessibility specifically related to macular degeneration (vision loss) (9). Single-cell ATAC-seqIn the coming years, ATAC-seq looks set to become a commonly used technique in single-cell analysis. Though ATAC-seq is not optimized for low cell numbers, modifications to the protocol have been made to accommodate this: microfluidics can be used to separate single nuclei and perform ATAC-seq reactions individually (10). An option with higher throughput is combinatorial cellular indexing, which uses barcoding to measure chromatin accessibility in thousands of individual cells. With this approach, there is the possibility to look at over 17,000 cells per experiment(10), although this technique is not truly a single-cell analysis. With single cell epigenomics the chromatin accessibility can be revealed cell by cell. Single-cell ATAC seq allows the identification of cell types and states for developmental lineage tracing. ATAC-seq will likely be a key component of comprehensive epigenomic workflows. Integration of whole-genome histone modification, DNA methylation, gene expression, and chromatin accessibility is becoming more common. Single experimental workflows to examine all these components together are on the horizon. ATAC-seq is well positioned to fulfil the chromatin accessibility portion of such workflows, due to its ease, speed, reliably, and multiplexing potential. Efficiency and limitationsMethods to assess open chromatin via next generation sequencing (NGS) have been in use for a number of years. The main advantage of ATAC-seq over existing methods is the simplicity of the library preparation protocol: Tn5 insertion followed by two rounds of PCR. After library preparation, the DNA is sequenced with NGS technology, and the number of reads for a region correlate with how open that chromatin is at a single nucleotide resolution. ATAC-seq requires no sonication or phenol-chloroform extraction like FAIRE-seq; no antibodies like ChIP-seq; and no sensitive enzymatic digestion like MNase-seq or DNase-seq. Unlike similar methods, which can take up to four days to complete, ATAC-seq preparation can be completed in under three hours (10,11). Another consideration in the ATAC-seq protocol is cell number. While the total number of cells defines library complexity, too few cells leads to under-transposition, while too many leads to over-transposition. It is therefore important to optimize cell number from the beginning: for human studies, 500–50,000 cells are recommended, but this can vary between species and cell type. The advantages of ATAC-seq include:
References
6. Corces, M.R., Granja, J.M., Shams, S., Louie, B.H., Seoane, J.A., Zhou, W., Silva, T.C., Groeneveld, C., Wong, C.K., Cho, S.W., et al. (2018). The chromatin accessibility landscape of primary human cancers. Science 362. 7. Prescott, S. L. et al. (2015). Enhancer Divergence and cis-Regulatory Evolution in the Human and Chimp Neural Crest. Cell 163, 68–83. 8. Lara-Astiaso, D. et al. (2014). Chromatin state dynamics during blood formation. Science 345, 943–949. 9. Wang, J., et al. (2018). ATAC-Seq analysis reveals a widespread decrease in chromatin accessibility in age-related macular degeneration. Nature Communications 9, 1364 10. Buenrostro, J. D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523,486–490 (2015). 11. Cusanovich, D. A. et al. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science 348, 910–4 (2015). External source
1 : Molecular biology techniques |
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