Imagine how long it would take to analyze the 37 trillion or so cells that make up the human body if you had to do it by hand, one by one! Still, single-cell analysis is crucial to gaining a comprehensive understanding of our biology. The cell is the unit of life for all organisms, and all cells are certainly not the same. Think about it: even though each cell contains the same DNA, some make up your skin while others build your bones; some of your cells might be super healthy while others could be headed down the road to cancer or Alzheimer’s disease.
So, it’s no surprise that many NIH-funded researchers are hard at work in the rapidly emerging field known as single-cell analysis. In fact, one team recently reported impressive progress in improving the speed and efficiency of a method to analyze certain epigenetic features of individual cells . Epigenetics refers to a multitude of chemical and protein “marks” on a cell’s DNA—patterns that vary among cells and help to determine which genes are switched on or off. That plays a major role in defining cellular identity as a skin cell, liver cell, or pancreatic cancer cell.
The team’s rather simple but ingenious approach relies on attaching a unique combination of two DNA barcodes to each cell prior to analyzing epigenetic marks all across the genome, making it possible for researchers to pool hundreds of cells without losing track of each of them individually. Using this approach, the researchers could profile thousands of individual cells simultaneously for less than 50 cents per cell, a 50- to 100-fold drop in price. The new approach promises to yield important insights into the role of epigenetic factors in our health, from the way neurons in our brains function to whether or not a cancer responds to treatment.
Despite rapid progress in single-cell analysis in recent years, Andrew Adey at Oregon Health & Science University, Portland, recognized two important technological gaps. One is the high cost of analyzing cells individually, which often limits researchers to examining relatively small numbers of cells. Another is that the available technologies for analyzing gene activity within thousands of individual cells were incompatible with methods to analyze the epigenetic marks on those cells.
Earlier work by the researchers showed it was possible to use a barcoding approach to examine how DNA is packaged into the strand-and-spool complex called chromatin  and also to sequence DNA from thousands of single cells at once . The key is to label individual cells not once but twice with a DNA barcode, and Adey and colleagues have a large library of unique ones at their disposal. Using a DNA sequencer to read the barcodes, researchers can specifically identify any individual cell mixed in among thousands of others.
Now, in the latest report published in Nature Biotechnology, Adey’s team shows that this approach also works for analyzing epigenetic features, specifically patterns of methylation. The term refers to the loss or gain of chemical methyl groups on DNA, which is important for determining the identity of a cell as it develops. Methylation patterns are also influenced by lifestyle and environmental factors.
After barcoding the cells, the researchers characterized their methylation profiles with bisulfite sequencing. In this established method, DNA is treated with a bisulfate salt, which chemically modifies the genome. By simply sequencing the treated DNA, it’s possible to infer the methylation status of DNA bases. Because the cells also displayed two DNA barcodes, the researchers found they could sequence thousands of cells at once and still identify each one individually.
To demonstrate how it works in practice, the researchers mixed three human cell lines together and showed that they could use the epigenetic profiles to differentiate among them. They also applied it to develop epigenetic profiles of brain tissue taken from the cortex of a mouse, and were able to identify distinct neural cell types. All told, they produced epigenetic profiles for more than 3,000 individual cells.
Now that they have proof of principle that the new method works, they’ll continue to explore its use in even more complex tissue samples, such as developing neurons. Ultimately, they want to combine this assay with others to explore additional epigenetic properties of cells and the role of those patterns in brain development, cancer, and other conditions. The exciting journey toward gaining a better understanding of the activity of individual cells and the inner workings of the human body continues, and this promising new approach will help to pick up the pace.
 Highly scalable generation of DNA methylation profiles in single cells. Mulqueen RM, Pokholok D, Norberg SJ, Torkenczy KA, Fields AJ, Sun D, Sinnamon JR, Shendure J, Trapnell, C, O’Roak BJ, Xia Z, Steemers FJ, Adey AC. Nature Biotech. 2018 April 9. [Epub ahead of print]
 Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Cusanovich DA, Daza R, Adey A, Pliner H, Christiansen L, Gunderson KL, Steemers FJ, Trapnell C, Shendure J. Science 2015 May 22;348(6237):910-914.
 Sequencing thousands of single-cell genomes with combinatorial indexing. Vitak SA, Torkenczy KA, Rosenkrantz JL, Fields AJ, Christiansen L, Wong MH, Carbone L, Steemers FJ, Adey A. Nat Methods. 2017 Mar;14(3):302-308.
Single Cell Analysis (NIH)
Epigenomics (National Human Genome Research Institute/NIH)
Adey Lab (Oregon Health & Science University, Portland)
NIH Support: National Institute of General Medical Sciences
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