Panel-level multilocus methylation quantification in native cell-free DNA by PCR-compatible sequential enzymatic processing
Year: 2026
Type: preprint
Abstract: 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.
Source: medRxiv
Authors: Cintia Celina Vaquer, Paula Alida Wetten, Clara García Samartino, Jorge Daniel Rodriguez, Ricardo Nicolás Manzino, Roberto Pérez Ravier, Agustina Renata Angeloni, Rodrigo Damián Militello, Alejandro Ledesma, Juliana Sesma, Yanina Agnella, Erika Gudiño, Carina Evelyn Fracchia Díaz, Rodrigo Ongay, Guillermo Sanguinetti, Agustín Correa, Melani Carlen, Walter Ramón Minatti, Gastón Andrés Vaschalde, Paula Valdemoros, Leandro Sarrió, Luis Mayorga, Victoria Bocanegra, Emanuel Martín Campoy
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7165515610