GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE328nnn/GSE328835/primary200OKOtherHomo sapiensOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE328835GEOGSEfalseIdentification of a G-quadruplex-forming cell-free DNA fragment as a biomarker for the precise diagnosis of hepatocellular carcinoma[cfDNA]Early detection of hepatocellular carcinoma (HCC) remains challenging, as the currently recommended surveillance strategy based on ultrasound combined with alpha-fetoprotein (AFP) is limited by suboptimal sensitivity and accessibility. Cell-free DNA (cfDNA) provides a minimally invasive avenue for cancer detection. However, most existing cfDNA-based approaches either perform unreliably in low-input samples or require analytically complex workflows. Here, we systematically profiled serum cfDNA from individuals with HCC and without HCC and identified a high-abundance tumor-associated single cfDNA fragment at the FAM230F genomic region. Integrative analysis of liver assay for transposase-accessible chromatin with sequencing (ATAC-seq) revealed consistent tumor-specific chromatin accessibility at this locus, suggesting a tumor-derived origin. Structural characterization further demonstrated enrichment of G-quadruplex (G4) features within the target sequence, which may increase resistance to serum nuclease degradation and promote its preferential retention in circulation. Based on these properties, we established a qPCR-based detection workflow with clinical accessibility. In a validation cohort independent of the discovery cohort, a ΔCt cutoff of 2 was selected by maximizing the Youden index within the same cohort. The assay showed a sensitivity of 94.5% and a specificity of 90.5% for distinguishing HCC from non-HCC. Collectively, our study identifies FAM230F as a structurally stable tumor-associated cfDNA fragment and establishes a simple and scalable qPCR-based assay for HCC detection, providing a practical framework for translating cfDNA fragment analysis into clinical biomarkers.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 sapiens