January 27, 2021—Ludwig Oxford’s Chunxiao Song and his team expand their suite of bisulfite-free sequencing methods to cover the direct and specific detection of all four modifications made to the DNA base cytosine.
Chemical modifications made to the DNA base cytosine play an important role in the regulation of gene expression across the genome. The base is chemically modified in four ways, with 5-methylcytosine (5mC) being the most common. The demethylation of 5mC by the TET family of enzymes results in the generation of the other three modifications: 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Each of these epigenetic modifications appears to have a distinct function. For example, 5mC is associated with repressed regions of the genome, whereas 5hmC is present in active ones.
The gold standard for detecting DNA methylation was, until recently, a method known as bisulfite sequencing. The method relies on a harsh chemical treatment that degrades most of the DNA sample being read. It also has the disadvantage of being an indirect detection method, which decreases sequencing quality. The new gold standard was established by Ludwig Oxford’s Chunxiao Song and his team with their development of a bisulfite-free method for 5mC sequencing, which they reported in Nature Biotechnology in 2019.
Named TAPS, their method directly detects modified cytosines, preserves DNA samples, is considerably more sensitive and produces sequencing data of higher quality. A lot higher, evidently: A biotechnology company Song and his colleagues established to commercialize this technology for liquid biopsies and other purposes was snapped up for $410 million by Exact Sciences in November 2020.
Good as it is, however, TAPS cannot distinguish between the different types of cytosine modifications. Other methods already exist that can do so, but they are indirect—using subtraction, for example, which entails measuring 5mC and subtracting this signal from a combined measurement of 5mC and 5hmC. In addition to the existing disadvantages of bisulfite and/or indirect detection strategies, subtraction methods also generate very noisy data that can be difficult to interpret. There has thus been a need in the epigenetics field for a subtraction-free method to specifically, directly and sensitively detect the four known cytosine modifications found in the genome.
And this is what Song and his colleagues have now accomplished. In this paper published in Nature Communications, Yibin Liu in Chunxiao Song’s lab and Zhiyuan Hu in Ahmed Ahmed’s lab at the University of Oxford report a suite of TAPS-related whole genome sequencing methods to directly detect all four modifications. They have named their methods TAPSβ (for 5mC), chemical-assisted pyridine borane sequencing (CAPS; for 5hmC), pyridine borane sequencing (PS; for 5caC and 5fC) and pyridine borane sequencing for 5caC (PS-c; for 5caC).
The new methods offer the research world powerful new tools to explore the distinct functions of cytosine modifications in gene regulation, including how their distribution is altered in diseases such as cancer.