Improving rare variant detection in next generation sequencing of liquid biopsies with a molecular barcoding technique that combines a novel primer structure and sophisticated bioinformatics methods

SummaryOur goal is to improve the sensitivityfidelityand efficiency of next generation sequencingNGSfor rare variant detection from liquid biopsy specimensExisting NGS techniques for liquid biopsy analysis suffer from several shortcomingssuch as compromises in sensitivity and precisioncumbersome workflowsor requirements for high nucleic acid inputs and or specialized instrumentationAs a consequencethe accessibility and clinical utility of this technology is currently limiteddespite its projected impact on the fields of oncology and precision medicine for the detection and monitoring of diseaseOur proposed solution overcomes these challenges through an innovative molecular barcodingMBCNGS strategy that leverages novel primer architectures and diagnostic facingassay tailored bioinformatics algorithmsOur innovative primer and barcode tag design simplifies overall NGS workflowreduces nucleic acid input requirementsand increases recovery of input templates to achieve the reliable and sensitive detection of lowlevelclinically actionable variantsA strand specific PCR protocol will be utilized that incorporates MBCs to support single molecule trackingerror correctionand accurate quantificationThe proposed method offers substantial improvements in the selective and efficient amplification of input templates and prevents unwanted off target effects such as primer dimersIn combination with these wet lab advanceswe will develop robust bioinformatics algorithms tailored to work hand in glove with the upstream MBC chemistriesThe use of MBCs will augment error correction and quantification strategies for the downstream informaticsThrough the datadriven development of rigorous probabilistic models for variant callingwe will achieveinmolecular detection sensitivity with high positive predictive valuePPVfor the clinical testing of liquid biopsy specimensWe propose the following two specific aimsAimDevelop a new molecular barcoding method for liquid biopsy samples using a novel temperaturetoggled hairpin looped primer and barcodeAimImprove the analysis of liquid biopsy samples using a robust bioinformatics analysis pipeline tailoredto PCR based molecular barcoded sequencing dataUltimatelythis proposal seeks to provide easy to usehigh performanceand kittable diagnostic products for the NGS of plasma liquid biopsies that can support routine and decentralized testing in a range of clinical laboratory settings with varying technical and operational sophistication Narrative This project offers novel methods that have been created to detect changes in blood samples to allow us to pinpoint these alterationsnamely cancer associated changes in the DNA of a patient s tumorThis information can help guide how to attack the cancer with more effective and less toxic therapiesWe ve achieved this goal by developing innovative technologies for molecular barcoding and analysis strategies that will improve nextgeneration sequencing and testing of patient s blood samples