New Sequencing Method Decodes DNA and Epigenetic Modifications Simultaneously
Researchers have developed a novel sequencing technique capable of differentiating between similar epigenetic modifications – 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) – while simultaneously preserving information about the underlying DNA sequence. This breakthrough addresses a long-standing challenge in epigenetics and opens new avenues for diagnostics and understanding gene regulation.
The Challenge of Epigenetic Mapping
The genetic code is often described as having four letters: adenine, guanine, cytosine, and thymine. However, modifications to these bases, particularly the addition of a methyl group to cytosine (creating 5mC), play a crucial role in gene expression and cellular identity. 5mC acts as an epigenetic marker, often silencing gene expression. Understanding these modifications is vital, but accurately detecting them has been complicated by the existence of 5hmC, a related modification formed when 5mC is oxidized.
Traditional methods, like bisulfite sequencing, struggle to distinguish between 5mC and 5hmC. Previous attempts to overcome this limitation often sacrificed genetic information. As chemist Shankar Balasubramanian noted in 2022, methods using deaminases, which convert cytosine to uracil but don’t act on 5hmC, “lose genetic information to gain the epigenetic information.”
Integrated Sequencing: A Novel Approach
The new method, dubbed “integrated sequencing,” developed by researchers at the University of Pennsylvania, overcomes this trade-off. The process begins by copying short DNA sequences into hairpin duplexes. Crucially, the cytosine, 5mC, and 5hmC on the newly synthesized strand are converted into analogs that resist deamination.
On the original, template strand of the hairpin, researchers selectively deaminate either only unmodified cytosine or both unmodified cytosine and 5mC. By sequencing both strands of the duplex, the team can recover both the complete DNA sequence and detailed information about its epigenetic markers.
Courtesy of Rahul Kohli
Benefits and Applications
According to graduate student Christian Loo, a key advantage of integrated sequencing is its ability to extract both sequence and modification information from the same molecule. “There are methods where you can computationally overlay different profiles, but if you have a method that can actually directly link information, that’s incredibly powerful,” he explains.
Chunxiao Song, who recently published a method for differentiating 5mC and 5hmC in single-cell DNA, describes the new technique as “a useful addition to and complement of existing copy-strand–based approaches for DNA modification analysis.”
The researchers envision applications in cell-free cancer diagnostics, where identifying rare mutant DNA molecules from cancer cells amidst a background of healthy cell DNA is critical. The epigenome of these mutant molecules could provide valuable information about the cancer’s origin.
Understanding 5-Methylcytosine
5-methylcytosine accounts for approximately 1%-6% of nucleotides in mammalian and plant genomes [1]. It is a modified base that adds an extra layer of heritable information to the DNA code, influencing the regulation of underlying genetic information. The spontaneous deamination of 5-methylcytosine results in thymine [3].
Laurel Oldach is a senior editor and life sciences reporter at C&EN.