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DMN-seq: DEMETER-Assisted 5-Methylcytosine Nicking Sequencing Profiling From Sub-Nanogram Cell-Free DNA

Lead Inventor: Chuan He

SUMMARY

More sensitive methylation profiling from scarce clinical samples is supported by a sequencing method that uses 5mC-specific glycosylase activity to detect methylated sites at single-base resolution and enrich hypomethylated DNA

The Unmet Need: Existing methylation methods struggle to profile hypomethylated DNA in scarce clinical samples

  • Bisulfite and related conversion methods can damage DNA or complicate mapping, while antibody-based enrichment tends to favor hypermethylated regions, lacks base-level resolution, and may require more input material. These limitations can hinder analysis of low-input samples and investigation of hypomethylated regions relevant to cancer biomarker discovery.

  • Sequencing-based epigenetic profiling is increasingly being applied to low-input clinical samples, including cell-free DNA, to discover and assess cancer biomarkers. Methods that combine precise methylation detection with enrichment of underexplored hypomethylated regions address an important opportunity in this area.

The Proposed Solution: Glycosylase-assisted nicking sequences 5mC sites and enriches hypomethylated DNA regions

  • The faculty inventor developed DMN-seq- a technique using 5mC-specific DNA glycosylase DEMETER (DME), which excises 5mC and nicks one DNA strand. The technology features specially designed adaptors and enzymatic cleanup capture DME-generated DNA ends to map and quantify 5mC at single-base resolution. The technique leverages repeated cleavage events removing methylated DNA to preferentially enrich hypomethylated regions.

  • Unlike conversion-based or antibody-enrichment approaches, the method provides base-level detection and supports profiling of hypomethylated regions. Validation includes synthetic DNA, bacteriophage λ-DNA, mouse embryonic stem-cell DNA, and a proof-of-concept study of paired colorectal cancer and adjacent tissue patient sampels. The method produced reproducible results with cell-free DNA inputs as low as 0.1 ng.

ADVANTAGES

  • Single-base 5mC resolution

  • Low-input sample compatibility

  • Reproducible methylation profiling

  • Hypomethylated-region enrichment

  • Low-background site detection

  • Dual methylation profiling workflows

APPLICATIONS

  • Cell-free DNA methylation profiling

  • Tumor-versus-adjacent-tissue methylation analysis

  • Profiling of promoter and enhancer hypomethylation tied to oncogenes and open chromatin

PUBLICATIONS