Simultaneous single-base resolution profiling of 5mC and 5hmC using BRIGHT-seq.

Cytosine modifications in DNA, particularly 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are key epigenetic marks involved in gene regulation and chromatin organization. Simultaneous and base-resolution detection of 5mC and 5hmC is essential for understanding their interplay in gene regulation. However, existing methods often rely on differential subtraction strategies, making direct and concurrent analysis within a single assay challenging. Here, we present the Base Replacement for Integrated Genome-wide Methylation and Hydroxymethylation Tracking Sequencing (BRIGHT-seq), a sequencing strategy that enables direct and simultaneous detection of 5mC and 5hmC at single-base resolution within the same DNA molecule. By integrating enzymatic and chemical treatments, BRIGHT-seq converts 5mC to adenine and 5hmC to thymine, enabling direct identification of both modifications without relying on differential subtraction. We demonstrate the applicability of BRIGHT-seq by mapping 5mC and 5hmC landscapes across human and mouse genomes. BRIGHT-seq provides a useful and high-resolution tool for epigenetic research, facilitating future studies on the roles of 5mC and 5hmC in gene regulation and disease.