Panel-level multilocus methylation quantification in native cell-free DNA by PCR-compatible sequential enzymatic processing
Vaquer, C. C.; Wetten, P. A.; Garcia Samartino, C.; Rodriguez, J. D.; Manzino, N. R.; Perez Ravier, R.; Angeloni, A. R.; Militello, R. D.; Ledesma, A.; Sesma, J.; Agnella, Y.; Gudino, E.; Fracchia Diaz, C. E.; Ongay, R.; Sanguinetti, G.; Correa, A.; Carlen, M.; Minatti, W. R.; Vaschalde, G. A.; Valdemoros, P.; Sarrio, L.; Mayorga, L.; Bocanegra, V.; Campoy, E. M.
Show abstract
DNA methylation is informative for liquid biopsy, but low template abundance, distributed methylation signals and workflow complexity limit implementation. Here we present Delta-HLD, a PCR-compatible methylation assay platform that quantifies methylation directly in native DNA through sequential hybridization, ligation and methylation-sensitive digestion. The assay co-reports methylation-dependent signals from multiple loci through a shared amplification architecture, generating a single panel-level PCR readout. We established the chemistry, optimized panel size and composition through model-guided experiments, and implemented the assay as a triplex qPCR workflow with per-sample internal process controls. Plasma proof-of-concept analyses showed discriminatory signal in CRC and proof-of-concept transferability to hepatocellular carcinoma. Additional platelet-retaining experiments identified a strategy to increase recovery of analyzable circulating templates while reducing genomic DNA recognition. Delta-HLD provides a compact PCR-compatible framework for low-input methylation analysis without base conversion.
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