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

## TL;DR

Active-Seq is a new method to profile unmodified DNA across the genome using low input DNA, enabling insights into cell-type-specific enhancers and disease profiling.

## Contribution

Active-Seq introduces a base-conversion-free, methyltransferase-directed method for isolating and profiling unmodified CpG sites.

## Key findings

- Active-Seq enables robust epigenomic profiling with as little as 1 ng of input DNA.
- The method enriches unmethylated enhancers that define cell-type identity.
- It is applicable to cell-free DNA and can be used for tumor-informed disease profiling in colorectal cancer.

## Abstract

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.

•Efficient, genome-wide profiling of unmodified DNA with as little as 1 ng input DNA•Unique targeting and enrichment of unmethylated enhancers that define cell type•Genome-wide, epigenomic profiling from as few as 70 million sequencing reads

Efficient, genome-wide profiling of unmodified DNA with as little as 1 ng input DNA

Unique targeting and enrichment of unmethylated enhancers that define cell type

Genome-wide, epigenomic profiling from as few as 70 million sequencing reads

Existing technologies for profiling DNA modifications focus on the detection of modified cytosine, which is typically associated with the inactivation of gene expression through promoter hypermethylation. However, DNA hypomethylation is a facet of early disease development, and unmethylated enhancers play an important role in defining cell-type identity. In this work, we sought to develop a robust method for genome-wide profiling of unmodified CpG sites.

Tosti et al. report a method for genome-wide, epigenomic profiling that enriches DNA containing unmodified CpG sites for analysis. The enzymatic approach does not damage DNA, nor does it rely on base conversion, and it allows profiling of cell-free DNA with as little as 1 ng of input material.