A better way to decipher DNA’s epigenetic code

Identifying disease may become easier with a new DNA sequencing method

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PHILADELPHIA – A new method for sequencing the chemical groups attached to the surface of DNA may be paving the way for better detection of cancer and other diseases, according to research from the Perelman School of Medicine at the University of Pennsylvania published yesterday in Nature Biotechnology.
These chemical groups mark one of the four DNA letters in the genome, and the differences in these marks along DNA control which genes are expressed or silenced. To detect disease earlier and with increased precision, researchers have a growing interest in analyzing DNA in settings in which there is a limited amount of material, such as the free-floating DNA that can be extruded from tumors into the bloodstream.
“We’re hopeful that this method offers the ability to decode epigenetic marks on DNA from small and transient populations of cells that have previously been difficult to study,” said co-senior author Rahul Kohli, MD, PhD, an assistant professor of Biochemistry, Biophysics and Medicine.
Researchers from Penn and elsewhere have investigated these DNA modifications over the last two decades to better understand and diagnose an array of disorders, most notably cancer. During this time, the major methods used to decipher the epigenetic code have relied on a chemical called bisulfite. While bisulfite has proven useful, it also presents major limitations — it is unable to differentiate the most common modifications on the DNA building block cytosine, and it destroys much of the DNA it touches, leaving little material to sequence in the lab.
“This technological advance paves the way to better understand complex biological processes such as how the nervous system develops or how a tumor progresses,” mentioned co-senior author Hao Wu, PhD, an assistant professor of Genetics. Kohli’s graduate student Emily Schutsky is first author on the study.
The new method described in the paper, entitled “Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase,” builds on the fact that a class of immune-defense enzymes, called APOBEC DNA deaminases, can be repurposed for biotech applications. Notably, the deaminase enzymes are able to achieve what bisulfite can do, without harming the DNA in the process.
“Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods,” the article abstract notes. “Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.”
Using the ACE-seq method, the team showed that determining the epigenetic code of one type of neuron used 1,000 times less DNA than required by the bisulfite-dependent methods. From this, the new method could also differentiate between the two most common epigenetic marks, methylation and hydroxymethylation.
“We were able to show that sites along the genome that appear to be modified are in fact very different in terms of the distribution of these two marks,” Kohli stated. “This finding suggests important and distinctive biological roles for the two marks on the genome.”

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