Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

5‐hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non‐enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein–DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single‐base resolution. We employ chemical oxidation to transform 5hmU to 5‐formyluracil (5fU), followed by the polymerase extension to induce T‐to‐C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T‐to‐C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU‐rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.