The discovery of epigenetic bases has revolutionised the understanding of disease and development. Among the most studied epigenetic marks are cytosines covalently modified at the 5 position. In order to gain insight into their biological significance, the ability to determine their spatiotemporal distribution within the genome is essential. Techniques for sequencing on “next‐generation” platforms often involve harsh chemical treatments leading to sample degradation. Third‐generation sequencing promises to further revolutionise the field by providing long reads, enabling coverage of highly repetitive regions of the genome or structural variants considered unmappable by next generation sequencing technology. While the ability of third‐generation platforms to directly detect epigenetic modifications is continuously improving, at present chemical or enzymatic derivatisation presents the most convenient means of enhancing reliability. This Review presents techniques available for the detection of cytosine modifications on third‐generation platforms.

The discovery of epigenetic bases has revolutionised the understanding of disease and development. Among the most studied epigenetic marks are cytosines covalently modified at the 5 position. In order to gain insight into their biological significance, the ability to determine their spatiotemporal distribution within the genome is essential. Techniques for sequencing on “next‐generation” platforms often involve harsh chemical treatments leading to sample degradation. Third‐generation sequencing promises to further revolutionise the field by providing long reads, enabling coverage of highly repetitive regions of the genome or structural variants considered unmappable by next generation sequencing technology. While the ability of third‐generation platforms to directly detect epigenetic modifications is continuously improving, at present chemical or enzymatic derivatisation presents the most convenient means of enhancing reliability. This Review presents techniques available for the detection of cytosine modifications on third‐generation platforms.