Panel-level multilocus methylation quantification in native cell-free DNA by PCR-compatible sequential enzymatic processing
- 1Instituto de Histologia y Embriologia de Mendoza (IHEM)-CONICET, Universidad Nacional de Cuyo (UNCUYO), Mendoza, Argentina; Facultad de Ciencias Medicas, Univer
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.
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BibTeX
@misc{vaquer2026panellevel,
title = {{Panel-level multilocus methylation quantification in native cell-free DNA by PCR-compatible sequential enzymatic processing}},
author = {C. C. Vaquer and P. A. Wetten and C. Garcia Samartino and J. D. Rodriguez and N. R. Manzino and R. Perez Ravier and A. R. Angeloni and R. D. Militello and A. Ledesma and J. Sesma and Y. Agnella and E. Gudino and C. E. Fracchia Diaz and R. Ongay and G. Sanguinetti and A. Correa and M. Carlen and W. R. Minatti and G. A. Vaschalde and P. Valdemoros and L. Sarrio and L. Mayorga and V. Bocanegra and E. M. Campoy},
howpublished = {medRxiv},
year = {2026},
month = jun,
doi = {10.64898/2026.06.11.26354897},
url = {https://www.medrxiv.org/content/10.64898/2026.06.11.26354897v2},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Panel-level multilocus methylation quantification in native cell-free DNA by PCR-compatible sequential enzymatic processing
AU - C. C. Vaquer
AU - P. A. Wetten
AU - C. Garcia Samartino
AU - J. D. Rodriguez
AU - N. R. Manzino
AU - R. Perez Ravier
AU - A. R. Angeloni
AU - R. D. Militello
AU - A. Ledesma
AU - J. Sesma
AU - Y. Agnella
AU - E. Gudino
AU - C. E. Fracchia Diaz
AU - R. Ongay
AU - G. Sanguinetti
AU - A. Correa
AU - M. Carlen
AU - W. R. Minatti
AU - G. A. Vaschalde
AU - P. Valdemoros
AU - L. Sarrio
AU - L. Mayorga
AU - V. Bocanegra
AU - E. M. Campoy
PY - 2026
DA - 2026/06/11
PB - medRxiv
DO - 10.64898/2026.06.11.26354897
UR - https://www.medrxiv.org/content/10.64898/2026.06.11.26354897v2
AB - 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.
ER -