2026 VAI Epigenetics Symposium

Sensitive detection of cancer-derived DNA fragments (ctDNA) within cell-free DNA (cfDNA) is critical for early cancer detection, treatment monitoring, and identification of minimal residual disease. A major challenge is that ctDNA often represents only a small fraction of total cfDNA. Recent advances in methylomic profiling, including 6-base sequencing with duet evoC, which distinguishes 5-methylcytosine (5mC) from 5-hydroxymethylcytosine (5hmC), enable highly sensitive ctDNA detection by analyzing methylation patterns across individual sequencing fragments rather than averaging signals at individual CpG loci.

Here, we present a comparative analysis of background error rates in fragment-level methylation analysis using the duet suite of biomodal methylation assays versus alternative methylation sequencing technologies. We observe error rates below 10⁻⁵ for both biomodal assays, compared with error rates exceeding 10⁻⁴ for alternative approaches. We further demonstrate how this improved analytical performance enables more sensitive detection of ctDNA. Finally, we apply these technologies to detect ctDNA in cfDNA samples from patients with stage I–IV colorectal cancer.

Together, our findings highlight the advantages of fragment-level methylation analysis for cfDNA-based applications and underscore the importance of assay performance in achieving high clinical sensitivity and specificity from limited cfDNA inputs.