Canadian Conference on Circulating Biomarkers (CCCB) 2026

Sensitive detection of cancer-derived DNA fragments (ctDNA) within cell-free DNA (cfDNA) in liquid biopsy is essential for identifying early-stage cancers, monitoring treatment response and minimal residual disease. A major challenge in these applications is detecting ctDNA, indicative of disease state, when this represents a small fraction of the total cfDNA. Advances in methylomic profiling, including 6-base sequencing with duet evoC which distinguishes 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), have enabled high-sensitivity ctDNA detection by analyzing methylation patterns across individual sequencing fragments (fragment level analysis) as opposed to more traditional analyses that average methylation levels at individual CpG loci.

We present a comparative analysis of the background error rates in fragment-level methylation analysis between the duet suite of methylation assays from biomodal and alternative methylation sequencing technologies, showing error rates below 10-5 for both biomodal assays compared to error rates above 10-4 for alternative technologies. We further show how this lower error rate can enable more sensitive detection of ctDNA, and present the application of these technologies to detecting ctDNA in cfDNA from patients diagnosed with stages I-IV colorectal cancer.

Our findings further emphasize the power of fragment-level analyses in cfDNA-based applications. They illustrate that it is critical to carefully consider analytical performance to achieve maximum clinical sensitivity and specificity from limited cfDNA samples.

Sensitive detection of circulating tumour DNA (ctDNA) within the total pool of cell‑free DNA (cfDNA) is crucial for early cancer diagnosis via liquid biopsy and for monitoring ctDNA levels during treatment and remission. This enables earlier assessment of treatment response, identification of minimal residual disease, and early detection of cancer. However, ctDNA typically represents only a minor fraction of overall cfDNA, posing significant challenges for conventional biomarker‑based detection methods. Recently, fragmentomics‑based approaches have shown promise, with studies demonstrating that analysis of fragment size distributions, 5′ end motifs, and nucleosome positioning near functional genomic sites can enhance sensitivity and specificity.

Advanced methylomic profiling methods, such as 6‑base sequencing with duet evoC, which distinguishes 5‑methylcytosine (5mC) from 5‑hydroxymethylcytosine (5hmC), further expand the analytical landscape. Integrating 5mC and 5hmC profiling with ctDNA detection strategies extends the spectrum of fragment end motifs and provides orthogonal data layers to fragment size and nucleosome positioning analysis. These multidimensional epigenetic signatures have the potential to improve the resolution and accuracy of ctDNA detection.

Here, we evaluate the impact of these additional epigenetic layers on ctDNA detection in liquid biopsy samples from healthy volunteers and patients with different stages of colorectal cancer. We observe distinct 5mC and 5hmC profiles and differences in fragmentomics metrics between healthy individuals and early‑ or late‑stage colorectal cancer patients. We also identify regulatory regions showing altered nucleosome positioning in cfDNA from colorectal cancer patients. Finally, we compare the performance of fragmentomics and epigenetic modalities, individually and in combination, for classifying colorectal cancer patients, demonstrating the utility of multiomic cfDNA datasets.