bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.07.14.738208

cf-Cabernet: Nick Repair Prior to End Repair Preserves Endogenous DNA Methylation Information in Cell-Free DNA Methylation Sequencing

Yuan, T.·Bai, Y.·Song, L.·Zhang, A.·Liu, Y.·Cao, Y.
View original source ↗Export metadata ↓

Abstract

Cell-free DNA (cfDNA) methylation profiling holds great promise for non-invasive cancer detection, yet accurate methylome analysis is compromised by DNA damage inherent to cfDNA. Standard library preparation workflows involve an end-repair step during which DNA polymerases can initiate synthesis from single-strand breaks (nicks), replacing endogenous methylated nucleotides with unmethylated nucleotides in the 3' direction and systematically erasing methylation information. This artifact is distinct from the terminal jagged-end effect and disproportionately affects cfDNA and FFPE DNA, which harbor abundant nicks. Here, we developed cf-Cabernet, which builds upon the Cabernet framework (Cao et al., 2023) -- an enzymatic methylation sequencing method featuring carrier DNA-assisted sample recovery and amplification-friendly post-conversion processing -- with the addition of a Taq DNA ligase-mediated nick repair step prior to end repair. Using matched cfDNA samples, we compared cf-Cabernet against standard EM-seq and WGBS. cf-Cabernet and EM-seq both substantially outperformed WGBS in alignment rate. Critically, while standard EM-seq exhibited globally reduced methylation levels compared to WGBS, cf-Cabernet yielded methylation levels concordant with WGBS. M-bias analysis revealed that EM-seq libraries showed persistently depressed methylation across the entire read length, whereas cf-Cabernet methylation recovered to the WGBS baseline beyond the terminal [~]40 bp jagged-end region. Nick-induced methylation erasure during end repair is a significant but previously underappreciated source of error in cfDNA methylation sequencing. cf-Cabernet effectively mitigates this artifact through pre-emptive nick ligation, enabling accurate methylome profiling from damaged DNA templates. This method is broadly applicable to cfDNA, FFPE DNA, and other clinical specimens where DNA integrity is compromised, providing a robust foundation for methylation-based liquid biopsy applications.

Explore related subjects

geneticsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Yuan, T., Bai, Y., Song, L., Zhang, A., Liu, Y., Cao, Y.. 2026-07-20. cf-Cabernet: Nick Repair Prior to End Repair Preserves Endogenous DNA Methylation Information in Cell-Free DNA Methylation Sequencing. https://doi.org/10.64898/2026.07.14.738208

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

OPA1 controls mitochondrial dysfunction-driven liver fibrosis in MASLD

Progressive hepatic fibrosis is the principal determinant of morbidity and mortality in metabolic dysfunction-associated steatotic liver disease and steatohepatitis (MASLD/MASH). Mitochondrial dysfunction is a hallmark of MASH, and the release of mitochondrial damage-associated molecular patterns (mito-DAMPs) from injured hepatocytes can promote fibrosis. However, how mitochondrial dynamics and quality control shape the fibrotic response in MASLD/MASH remains unclear. Here, through large-scale genomic analyses of mitochondrial genes governing mitophagy, fusion and fission in human MASLD, with a power-equivalent sample size of approximately 700,000 individuals, we identify a strong association between hepatic fibrosis and the mitochondrial fusion factor dynamin-like GTPase optic atrophy 1 (OPA1). OPA1 transcripts and protein abundance in the liver epithelium were progressively dysregulated with advancing fibrosis. In mice, hepatocyte-specific OPA1 loss alone was sufficient to induce hepatic stellate cell activation and fibrosis in zone 3, promoted the release of mito-DAMPs into the circulation and exacerbated fibrosis in experimental MASH. These findings identify OPA1 as a central regulator of the hepatic fibrotic response and connect defective mitochondrial homeostasis to mito-DAMP release, hepatic stellate cell activation and fibrosis in MASLD.

genetics↗
bioRxiv2026

Mechanism-selective deep mutational scanning distinguishes ERCC2 disease phenotypes

Pathogenic ERCC2 variants cause xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or both, yet variant effect scores are usually interpreted only as measures of pathogenicity rather than of which disease mechanism is disrupted. XPD, the ERCC2-encoded TFIIH subunit, functions in both nucleotide excision repair and transcription. Using yeast complementation deep mutational scanning, we measured the effects of nearly all XPD amino acid substitutions. The assay was mechanism-selective: it preferentially reported transcription-associated function, with pronounced intolerance at the p44 interface, whereas many substitutions affecting DNA binding and helicase activity retained near-wild-type fitness. Accordingly, TTD variants had much lower fitness than XP variants. Computational predictors discriminated pathogenic from benign variants similarly across phenotypes, but the DMS distinguished XP from TTD variants better than all 73 predictors tested. Phenotype-specific ACMG/AMP calibration provided evidence in both directions for TTD but mainly pathogenic evidence for XP. Thus, the selectivity of functional assays, often viewed as a limitation, can reveal disease mechanisms and support phenotype-aware variant interpretation.

genetics↗
bioRxiv2026

Temporal control of mitochondrial mutagenesis reveals the fate of mtDNA mutations with age

Mutations in the mitochondrial genome (mtDNA) play a critical role in the aging process and a wide variety of age-related diseases. However, it remains unclear when the mutations that drive physiological decline arise. To answer this question, we generated a new mouse model in which mitochondrial mutagenesis can be confined to a defined window of time. Surprisingly, we found that mutations that arise during the first two months of life are sufficient to drive a wide variety of age-related pathologies, and that the severity of this pathology is broadly regulated by distinct, tissue-specific selective pressures that control the fate of mtDNA mutations with age. Further, we found that selection against deleterious variants can be modulated by manipulation of mitochondrial fusion in vitro and in vivo. These observations raise the possibility that in some tissues, the pace of aging is pre-determined by events that occur early in life and that interventions targeting mitochondrial fusion may be able to slow down or reverse the expansion of these pathogenic variants. These results carry far-reaching implications for strategies aimed at preventing or delaying age-related decline.

genetics↗