Unnatural cytosine analogs potentiate a customizable, enzymatic method for integrated epigenetic and four-base genetic sequencing

However, standard methods for profiling DNA modifications (e.g., bisulfite-sequencing) rely on selective C-to-T conversions, hindering the simultaneous examination of both genetic and epigenetic information. Here, we introduce Integrated-Sequencing, which provides high-fidelity mapping of DNA modifications while preserving the native four-base genetic code in single molecules.

Integrated-Seq leverages the synthesis of a tethered copy strand with unnatural cytosine analogs that resist enzymatic conversion, combined with a novel DNA deaminase-helicase fusion that drives selective C-to-T conversion of natural cytosines in the original template strand. We demonstrate that Integrated-Seq is compatible with customizable enzymatic readouts to parse 5-methylcytosine and 5-hydroxymethylcytosine, and that preservation of the original four-base genetic code markedly improved enrichment, facilitating analysis of targeted genomic regions.