Early cancer detection requires high-performing biomarkers and effective tools to enable early intervention. Telomeres, located at the terminal of linear chromosomes, affect genome integrity and cell immortalization. At the very end of the telomere is a 3' overhanging guanine-rich tail (G-tail) of which shortening is closely related to cell divisions. However, G-tail DNA cannot be detected with conventional sequencing technologies. Here we develop a high-preservation technology to quantify

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... eres in circulating cell-free DNA and show that short G-tail DNA was nearly 20-fold higher in hepatocellular carcinoma patients with blood collected at diagnosis than controls and was also strongly correlated with poorer survival. We then used an independent prospective population cohort to show that the abundance of circulating short telomere G-tail increased with time approaching cancer diagnosis. These results indicate that circulating short-telomere G-tail, measured using our novel bilateral single-strand sequencing method, can effectively detect early and aggressive hepatocellular carcinoma.