Targeting the Unmethylated Genome: A New Approach to Epigenetic Profiling
A novel enzymatic platform for epigenomic analysis has been developed, requiring only a fraction of the sequencing depth typically required by traditional approaches.
Researchers from Tagomics, the University of Birmingham, and the Van Andel Institute have developed “Active-Seq”, a novel enzymatic platform for epigenomic analysis that requires just a fraction of the sequencing depth of traditional approaches. Published in Cell Reports Methods, the study demonstrates genome-wide epigenomic profiling with DNA inputs in the (sub)nanogram range. The multi-institutional study, led by Tagomics, demonstrates how a focus on unmethylated DNA could revolutionize epigenetic analysis, particularly for liquid biopsy samples.
A story of two parts: methylated and unmethylated DNA
Unsurprisingly, current DNA methylation technologies focus on detecting DNA methylation. This modification is typically associated with gene silencing. The unmethylated regions of the genome tell the opposite, equally important story of our active, accessible DNA. For example, unmethylated enhancers define cell-type identity, and DNA hypomethylation is a characteristic feature of early cancer development.
We developed Active-Seq to target the analysis of these understudied genomic features, and to avoid the expense and sequencing redundancy associated with existing genome-wide, base-converting approaches for studying DNA methylation.
We tailored Active-Seq for application in liquid biopsy testing, focusing efforts on a workflow that was simple to implement, non-damaging towards DNA, and robust and reproducible with DNA input amounts on the nanogram scale, typical of liquid biopsy samples.
A streamlined enzymatic approach
We developed a single-tube workflow that combines enzymatic DNA tagging, enrichment of unmethylated DNA, and library preparation. The method uses a repurposed bacterial methyltransferase enzyme and a synthetic cofactor analog to enable capture and enrichment of unmethylated DNA fragments.
The study showed that the workflow is highly efficient and compatible with DNA inputs as low as 1 ng, a critical threshold for analyzing cell-free DNA (cfDNA) from liquid biopsies. We benchmarked Active-Seq against whole genome bisulfite sequencing and showed accurate mapping of genomic methylation levels with Active-Seq using only a fraction of the sequencing reads.
Finally, we showed the application of Active-Seq for biomarker discovery using tissue samples from cancer patients, and for the epigenomic profiling of cfDNA from cancer patients.
The key findings of the paper were:
- Active-Seq achieves approximately 160-fold enrichment of unmethylated DNA over background, with workflow efficiency of 60–80% DNA recovery.
- Genome-wide, epigenomic profiles can be generated using around 1/10th of the sequencing reads required for comparable base-converting technologies.
- Enriched, unmethylated regions of the genome overlap with promoters, enhancers and other transcriptionally-active genomic elements.
- Comparative studies using Active-Seq enable genome-wide biomarker discovery.
- Epigenomic profiling of cancer patient cfDNA suggests Active-Seq will find application in clinical biomarker discovery and cancer diagnostics in the future.
Robust, genome-wide epigenomic profiling
We showed that Active-Seq provides an information-rich, genome-wide epigenetic profile that is unique amongst methylation profiling technologies in its ability to target the 20–30% of CpG sites in the genome that are unmethylated. This focus on unmethylated regions allows cost-effective epigenomic profiling with a robust readout that anticorrelates to the methylation levels of the gold standard, whole genome bisulfite sequencing.
Active-Seq is highly reproducible, with correlation coefficients of 0.91–0.96 between technical replicates. We showed that it can be applied to liquid biopsy, fresh-frozen tissue and FFPE tissue samples.
The unmethylated genome includes cell-type-defining enhancers and partially methylated domains that see reduced DNA methylation as a result of rapid tumor cell division. These biological features, amongst others, provide key diagnostic information that complements the focus of existing methylation-based approaches.
We successfully applied Active-Seq to tumor and normal adjacent tissue from a small cohort of colorectal cancer patients and were able to identify tens-of-thousands of tumor-associated DNA methylation changes. The regions identified correlated with known tumor biology.
Using cfDNA from colorectal cancer patients, we were able to identify cancer cases using the signal derived from regions of the genome identified as hypomethylated in solid colorectal tumours.
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Our study builds on the pioneering development of this approach by Kriukienė et al., by significantly improving the efficiency of the enrichment process. This transforms the utility of the technology from a research tool to one that can be applied in the clinic on cfDNA extracted from patient samples.
This study focuses on the development and technical validation of the Active-Seq platform. Early results show the promise of the platform applied to clinical samples, yet patient sample numbers in the current study are not sufficient to build the case for clinical validation. This is the objective of our current work.
Delivering a complete picture of the epigenome at scale
With a significantly reduced sequencing burden over the gold-standard genome-wide methylation profiling technologies, Active-Seq opens new avenues for epigenetics as a research tool, and in clinical applications. Our immediate next steps include expanding the colorectal cancer cohort to a pilot clinical study, to validate the platform's utility for early disease detection.
The method's compatibility with liquid biopsy samples and its ability to enrich both hypermethylated promoters and hypomethylated domains make it uniquely suited for comprehensive cancer profiling.
Through Tagomics’ ActiveAce platform, we envision Active-Seq becoming a foundation for streamlined, multiomic profiling from liquid biopsy samples. The technology provides access to genetic, epigenetic and fragmentomic information in cfDNA, allowing us to build a comprehensive picture of patient wellbeing from a simple blood test.
Reference: Tosti L, Mould C, Gatehouse I, et al. Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment. Cell Rep Meth. 2025;5(10):101187. doi: 10.1016/j.crmeth.2025.101187
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