Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment.

We present "Active-Seq" (azide click tagging for in vitro epigenomic sequencing), a base-conversion-free technology that enables the isolation of DNA containing unmodified CpG sites using a mutated bacterial methyltransferase enzyme and a synthetically prepared cofactor analog. Active-Seq is a robust epigenomic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation and compatible with DNA input quantities as low as 1 ng. We establish a baseline for the performance of Active-Seq using model DNA oligos and further validate it against gold-standard whole-genome bisulfite sequencing data. We show robust performance of the platform across tissue-derived DNA and demonstrate enrichment of DNA at unmethylated, cell-type-specific marker regions of the epigenome, laying the foundation for the future application of this technology in tissue deconvolution applications. Finally, we apply the technology to cell-free DNA samples, outlining an approach for tumor-informed disease profiling in patients with colorectal cancer.