Improved circulating tumor DNA profiling by simultaneous extraction of DNA methylation and copy number information from methylated DNA sequencing data.

Cell-free DNA (cfDNA) analysis offers a powerful, minimally invasive approach to improve cancer care by measuring tumor-specific genomic and epigenetic alterations. Here, we demonstrate the versatility of MeD-seq, a methylation-dependent sequencing assay, for comprehensive cfDNA analysis, including DNA methylation profiling, chromosomal copy number (CN) alterations, and tumor fraction (TF) estimation. MeD-seq-derived CN profiles and TF estimates from 38 colorectal cancer patients with liver metastases (CRLM) and 5 ovarian cancer patients were highly comparable to shallow whole-genome sequencing (sWGS), validating our approach. For 120 CRLM patients, we used MeD-seq CN and TF information in an improved differential methylation model, which detected additional significantly Differentially Methylated Regions (DMRs) correlating with TF estimates. Using the identified DMR sets, we were subsequently able to distinguish healthy blood donors from CRLM patients with low amounts of circulating tumor DNA (ctDNA) as well. These findings establish MeD-seq as an affordable platform for tumor-agnostic detection of cancer-specific signals in plasma. This methodological framework provides a foundation for clinical applications requiring sensitive ctDNA detection from limited sample material, including treatment response assessment and minimal residual disease monitoring.