SRP176482 PRJNA513381 GSE124775 Multiplex 5mC Marker Barcode Counting (MMBC-seq): A sensitive detection method for cell-free DNA methylation The detection of hypermethylation markers on cell-free DNA (cfDNA) in biological fluids is a promising and non-invasive approach for early diagnosis and monitoring of human diseases. However, it is challenging to detect hypermethylation markers in a high-throughput, sensitive, and cost-effective manner. Here we presented a multiplex 5-methylcytosine marker barcode counting (MMBC-seq) technique and reported its clinical application for cfDNA from peripheral plasma samples. We identified an MMBC cancer detection panel and developed a scoring system to differentiate cancer versus healthy controls. In a multiple-cancer case-control study, the panel achieved a sensitivity and specificity of 80.2% and 95.7% respectively (AUC 0.906, 95% CI 0.846-0.948). The results suggest that MMBC-seq has great potential to realize non-invasive, flexible and clinically applicable cancer detection. Overall design: A total of 58 healthy individuals, 121 cancer patients, 19 patients with benign disease from China. GSE124775