Non-destructive methylation sequencing enables concurrent detection of genetic and epigenetic variation in FFPE and cfDNA samples

Comprehensive tumor characterization increasingly requires both genomic and epigenomic information, yet current methylation profiling methods, such as bisulfite-based workflows, limit sensitivity and accuracy due to DNA degradation and reduced sequence complexity. To address these constraints, we developed an improved positive-conversion chemistry that directly converts methylated cytosines while preserving unmethylated ones to enable simultaneous detection of cytosine modifications and genetic variants from a single NGS library. This approach is designed to support oncology research applications where high accuracy from limited or damaged material, such as FFPE tissue and cell-free circulating tumor DNA (ctDNA), is essential. To assess performance, FFPE, fresh-frozen, and cfDNA samples were processed using an optimized oxidation-reduction workflow. Libraries were sequenced on standard short-read platforms, and methylation accuracy, F1 scores, CNV concordance, and tumor-normal methylation contrast were evaluated. Performance was compared to whole-genome sequencing (WGS) controls and conventional bisulfite and enzymatic methylation sequencing methods.The chemistry demonstrated efficient reduction of modified cytosines, enabling high-confidence methylation calling with low false positive rates. Sequence complexity was maintained, allowing robust SNV and CNV detection from the same library. FFPE samples exhibited improved CpG coverage, fewer sequence artifacts, and improved library complexity compared to enzymatic methylation sequencing. cfDNA libraries generated from as little as 1 ng yielded reliable global methylation profiles suitable for tissue-of-origin analyses. The nondestructive nature of the chemistry preserved cfDNA fragments, opening the door to fragmentomics analyses. In tumor-normal comparisons, there was clear resolution of differentially methylated regions (DMRs), supporting more precise identification of tumor-specific epigenetic alterations. This enhanced positive-conversion chemistry enables unified genomic and epigenomic analysis from a single assay, making it highly suited for oncology research applications such as tumor classification, biomarker discovery, minimal residual disease (MRD) assessment, and liquid biopsy. Its gentle conditions, compatibility with degraded FFPE DNA and low-input cfDNA, and ability to capture both methylation and variants position it as a powerful tool for advancing translational research and precision oncology.