Epigenomic profiling of active regulatory elements by enrichment of unmodified CpG dinucleotides

Current approaches for the study of DNA methylation and other modified bases focus on the modified fraction of the genome and are particularly well-suited to the detection of DNA hypermethylation. However, the study of hypomethylation (loss of DNA methylation), which is typically associated with markers of active chromatin, has been largely overlooked, in part, due to the lack of a suitable methodology. We present an enrichment-based and bisulfite-free approach for epigenomic profiling named "Active-Seq" (Azide Click Tagging for In Vitro Epigenomic sequencing) that achieves genome-wide profiling of DNA, by enriching for non-modified CpG sites using a mutated methyltransferase enzyme. We show that the genomic regions enriched by Active-Seq overlap with promoters, enhancers and partially methylated domains, all of which have had their methylation status linked to the development and progression of diseases. Active-Seq is a fast epigenetic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation. The enzymatic chemistry is non-damaging toward the DNA which is critical for working with low concentration DNA input and will facilitate the future development of multiomics assays. We demonstrate robust and reproducible performance of Active-Seq using low DNA input in cell lines, liquid biopsies (cell-free DNA, cfDNA) and formalin-fixed paraffin embedded (FFPE) tissue.