Background: Multiomic strategies are increasingly used in oncology research by integrating genomic, epigenomic, transcriptomic, proteomic, and fragmentomic information. The combined assessment of DNA methylation and somatic variants in cell-free DNA (cfDNA) is particularly informative for identifying tissue- and tumor-specific signals. However, many current methylation profiling methods require harsh chemical treatments that degrade DNA or limit compatibility with standard sequencing workflows.

Methods: Using a novel, non-destructive approach that directly converts 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to thymine through sequential oxidation and reduction, thereby preserving DNA integrity. A streamlined workflow with improved 5mC conversion and library complexity was evaluated on cancer-derived samples using whole genome sequencing (WGS) and hybrid capture panels. Analyses focused on methylation profiling, somatic variant detection, and compatibility with multimodal sequencing workflows.

Results: Libraries exhibited high complexity and uniform genome-wide coverage, enabling concurrent assessment of epigenetic and genetic features from a single input. Because the chemistry converts only modified cytosines, the vast majority of bases remain unaltered, supporting improved base quality and accurate variant calling. Tissue- and tumor-specific differentially methylated regions (DMRs), as well as C>T mutations, were reliably detected. Libraries also performed robustly in target enrichment workflows using standard hybrid capture panels, yielding deep, uniform coverage across DMRs and somatic variants in the same assay.