Mutation sequencing of cell-free DNA (cfDNA) in liquid biopsy is crucial for tumor precision medicine, resistance profiling, and clinical decision-making. However, cfDNA mutation profiling in blood samples remains challenging due to its low abundance and high background noise. Here, we develop an Enzymatic Cleavage-directed Single Nucleotide Variant Sequencing (EC-SNV-Seq) assay technology for multiple cfDNA mutation detections. The EC-SNV-Seq assay integrates Argonaute-mediated cleavage and stem-loop DNA-initiated cascade-PCR amplification, achieving a sensitivity of 0.01% variant allele frequency. As a proof of concept, we apply the EC-SNV-Seq assay to detect multiple cfDNA mutations in KRAS, EGFR, and PIK3CA genes with single-nucleotide resolution. We further validate that EC-SNV-Seq detects early-stage multi-cancer and identifies single-nucleotide variants in a single patient plasma sample. Lastly, we evaluate its feasibility for multi-cancer early detection in population-scale screening using pooled plasma samples. The EC-SNV-Seq assay can enable highly sensitive and specific identification of low-frequency mutations, facilitating early cancer diagnosis and personalized treatment strategies.
