A fast, scalable, cost-effective, single-stranded library protocol for cell-free DNA

Project Summary Liquid biopsy is a potentially powerfulnon invasive approach for detectingclassifying and monitoring tumors by capturing cell free DNA molecules found circulating in blood and other bodily fluidsAny next generation sequencingNGSbased approach requires an efficient protocol for converting cell free DNA into a sequencing libraryNearly all currently available NGS library preparation methods target exclusively double stranded DNAHoweverthe benefits of a single stranded DNA library approach are well describedhigher efficiency and fidelityless length biasrecovery of nicked double stranded fragmentsand retention of the native ends of each moleculei eno DNA end repairDespite the benefitssingle stranded library generation has not been widely adopted because the only existing protocols are expensivecomplicatedand timeconsumingClaret Bioscience has developed an innovative new single stranded approachSRSLYthat is fastefficientsimple to followand cost effectiveThe protocol is carried out in a single tube and can convert cell free DNA into a sequence ready product in less than three hoursWe propose to develop the SRSLY protocol for clinical analysis by optimizing the reaction conditions for cell free DNA isolated from blood plasmaWith data from our sequencing librarieswe will investigate the added utility of retaining the native ends of cell free DNA molecules for reconstructing higher resolution nucleosomemapsas well as recovering populations of short cell free DNAeither nicked fragments lost to other methods or linker molecules thought to be associated with regulatory elements with potential significance in disease processesIn collaboration with medical oncologists at UCSFwe will analyze cell free DNA from a study of GI cancer patients through their course of treatment and integrate our sequencing based metrics with other metrics of tumor progression to learn the utility of our method in a clinical settingWe aim to develop a scalablecost effective protocol that supports all NGS liquid biopsy analyses in current use and provide data suitable for new discovery Project Narrative In patients with tumorsDNA fragments collected from blood plasma often include a small fraction of fragments that derive from the tumor tissue itselfproviding an invaluable opportunity to monitor tumor progression and patient health non invasivelyClaret Bioscience is developing an innovative new approach to convert cell free DNA fragments into next generation sequencingNGSlibrariesOur approach is fastmore efficient than existing methodsand retains important biological information that is lost or altered by existing approachesOur goal is to optimize this cost effective NGS library preparation method for low amounts of cell free DNA for use in liquid biopsy assays