Panel-level multilocus methylation quantification in native cell-free DNA by PCR-compatible sequential enzymatic processing

DNA methylation provides informative signals for liquid biopsy, but low template abundance, distributed signals across loci and conversion-dependent processing can complicate implementation in low-input samples. Here we present Delta-HLD, a PCR-compatible platform that quantifies methylation directly in native DNA through sequential hybridization, ligation and methylation-sensitive digestion. By encoding locus specificity at the hemi-probe hybridization and ligation stage, Delta-HLD allows methylation-dependent products from multiple loci to converge on a shared amplification and detection system and contribute to a single panel-level readout. We established the assay chemistry, optimized panel composition through model-guided experiments and implemented the platform in qPCR and ddPCR, including a triplex qPCR format with per-sample digestion and amplification controls. Proof-of-concept plasma analyses showed discriminatory signals in colorectal cancer and supported transfer of the same assay logic to hepatocellular carcinoma. The assay was further adapted for cfDNA analysis following platelet-retaining processing, preserving cfDNA-associated signal while reducing genomic DNA recognition. Delta-HLD provides a compact native-DNA platform for multilocus methylation analysis in low-input settings without base conversion.