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A collaboration between Prof Ahmed's group at the Nuffield Dept of Women's & Reproductive Health and Dr Chunxiao Song’s lab at the Ludwig Institute for Cancer Research, has established new sequencing methods for distinguishing DNA modifications. The paper: "Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution" has been published in Nature Communications.

The chemical structures of the four modified cytosine bases in DNA: 5mC, 5hmC, 5fC and 5caC.

Chemical modifications made to the DNA base cytosine play an important role in the regulation of gene expression across the genome. Cytosine can be chemically modified in four ways, with 5-methylcytosine (5mC) being the most common. Demethylation of 5mC by the TET family of enzymes results in the stable intermediates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). From what has been discovered so far, these modifications appear to have distinct functions. For example, 5mC is associated with repressed regions of the genome whereas 5hmC is present in active ones. However, to study these modifications further, robust sequencing methods are needed that can detect each of these four modifications specifically.

The traditional gold standard method for detecting DNA methylation is bisulphite sequencing. However, this relies on a harsh chemical treatment that degrades most of the DNA sample and is an indirect detection method, which decreases sequencing quality. Recently, a bisulphite-free method called TAPS has been developed by Ludwig Oxford’s Song lab, which has the advantage of preserving more of the DNA, increasing sensitivity, and directly detecting modified cytosines for improved DNA sequencing quality.

Despite its advantages, TAPS cannot distinguish between the different types of cytosine modifications. Other methods already exist that can do so but these use subtraction, for example, measuring 5mC and subtracting this signal from a combined measure of 5mC and 5hmC to obtain 5hmC levels. In addition to the disadvantages of using bisulphite and/or indirect detection strategies, these subtraction methods also need higher sequencing depths and generate very noisy data that can be difficult to interpret. New subtraction-free methods are therefore needed to specifically, directly and sensitively detect these four cytosine modifications in the genome.

In this paper published in Nature Communications, Dr Yibin Liu from Dr Chunxiao Song’s lab (Ludwig Oxford) and Dr Zhiyuan Hu from Professor Ahmed Ahmed’s lab (Weatherall Institute of Molecular Medicine and Nuffield Department of Women’s and Reproductive Health, University of Oxford) have developed a suite of TAPS-related whole genome sequencing methods for specifically detecting 5mC, 5hmC, 5fC and 5caC. They have named these 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).

With these new methods, the research community is now armed to tackle more of the questions about the distinct and important functions of cytosine modifications in the genome and how their distribution is altered in diseases such as in cancer.