Identifying the right amount of therapy – no more and no less – for patients with early stage cancer remains a challenge because there is no reliable method by which to separate those with microscopic residual disease after systemic or local therapies, from those without it. Current imaging can barely detect a mass of 1 million cells, while it takes just one cell to spawn new tumors that, by the time they are detected, are often incurable. Early detection of minimal residual disease (MRD) could give patients who need further treatment a chance at a cure, and prevent over-treatment of others. Despite its promise, MRD detection based on technologies like digital PCR that detect a single tumor marker at a time has inadequate ability to detect residual cancer at early stages. Next generation sequencing (NGS) can track many mutations simultaneously; however NGS requires extensive corrections using molecular barcoding to reduce noise and detect low-level mutations. This requirement invariably diminishes NGS throughput and increases expense. Currently, for NGS it is either sequencing depth or breadth, but not both. Here we propose to refine and apply a transformative technology that enables highly sensitive tracing of MRD in blood despite limited cfDNA material, while also retaining NGS throughput (breadth) and depth. We recently developed NaME-PrO, a simple and powerful technology that enables NGS to track extremely low-level mutations in circulating DNA. NaME-PrO utilizes a nuclease guided by probes to thousands of DNA targets, to render WT sequences non-amplifiable thereby allowing mutation– containing sequences to amplify and be sequenced with few reads as if they were high abundance mutations. To track MRD in blood, we first create a tumor fingerprint for each patient using whole exome sequencing of the primary tumor to define 30-100 tumor-specific clonal mutations and encompassing truncal mutations. These will be tracked in cfDNA using NaME-PrO-enhanced NGS. NaME-PrO will be combined with molecular barcoding (qNaME-PrO) to enable quantification of the original mutation fraction with few sequencing reads for the patient-specific mutations tracked and elimination of errors. We will (a) optimize and test the use of molecular barcoding in conjunction with NaME-PrO mutation enrichment for quantification of original mutation abundance; (b) Perform exome sequencing to derive mutational tumor fingerprints; then follow fingerprints in plasma and serial dilutions in WT plasma to determine the lowest limit of quantitative detection; and (c) perform a preliminary assessment of the prognostic ability of MRD in melanoma patients. If the project is successful, it will be followed by practice- changing clinical studies. The proposed method is anticipated to provide a high negative predictive power, as one of the main advantages. This could eventually enable `watchful waiting' strategies for some patients currently treated unnecessarily, thus reducing morbidity a...