Project Summary Oncologists have a great need for improved DNA sequencing technology because sequencing the DNA of cancer cells can provide valuable information about the specific mutations driving the disease and inform/guide treatment decisions. Additionally, sequencing healthy tissue can provide information about an individual's genetic risk factors for cancer. One area of particular interest is repetitive DNA sequences called microsatellites, which undergo a high level of polymorphism, defined as microsatellite instability (MSI). MSI is a type of genetic alteration that is often found in many cancer cells, especially in certain types of tumors, such as colorectal cancer. The detection of MSI is important for the diagnosis and management of cancer patients. For example, MSI-high tumors are more responsive to immunotherapy, a type of cancer treatment that boosts the body's immune system to attack cancer cells. Therefore, accurate microsatellite sequencing is crucial for oncologists to make informed treatment decisions for their patients. Currently available DNA sequencing technologies, such as Sanger sequencing, next-generation sequencing (NGS), and single-molecule sequencing (SMS), all have limitations when it comes to accuracy, cost, and scalability. During this Phase I SBIR program, Electronic BioSciences, Inc. (EBS) will be developing a “Fourth Generation” DNA sequencing technology that enables de-novo, ultra-high accuracy sequencing, specifically targeting microsatellites for oncology applications. This project directly leverages recent advances in nanopore-based sequencing and single-molecule strand control/manipulation to enable the direct, continuous re-sequencing of individual DNA molecules, which will enable unprecedented levels single-molecule sequence coverage and subsequent high-accuracy sequence determinations during sample profiling/screening. The technology developed during this project will enable the confident detection of MSIs with greater precision than any currently available methods. The system will also be automated and scalable for eventual high-throughput sequencing. Further, to validate the sequencing improvements achieved during this project, the developed technology will be benchmarked against currently available approaches. Ultimately, this project has the potential to significantly advance the fields of oncology and cancer genomics by enabling the accurate and efficient sequencing of DNA, including challenging microsatellite regions, which can serve as critical biomarkers for cancer diagnosis and prognosis, and guide the development of effective therapeutic interventions.