Research Project
9557901
Summary Our goal is to improve the sensitivity, fidelity, and efficiency of next-generation sequencing (NGS) for rare variant detection from liquid biopsy specimens. Existing NGS techniques for liquid biopsy analysis suffer from several shortcomings, such as compromises in sensitivity and precision, cumbersome workflows, or requirements for high nucleic acid inputs and/or specialized instrumentation. As a consequence, the accessibility and clinical utility of this technology is currently limited, despite its projected impact on the fields of oncology and precision medicine for the detection and monitoring of disease. Our proposed solution overcomes these challenges through an innovative molecular barcoding (MBC) NGS strategy that leverages novel primer architectures and diagnostic-facing, assay-tailored bioinformatics algorithms. Our innovative primer and barcode tag design simplifies overall NGS workflow, reduces nucleic acid input requirements, and increases recovery of input templates to achieve the reliable and sensitive detection of low- level, clinically-actionable variants. A strand-specific PCR protocol will be utilized that incorporates MBCs to support single-molecule tracking, error correction, and accurate quantification. The proposed method offers substantial improvements in the selective and efficient amplification of input templates and prevents unwanted off-target effects such as primer-dimers. In combination with these wet-lab advances, we will develop robust bioinformatics algorithms tailored to work hand-in-glove with the upstream MBC chemistries. The use of MBCs will augment error correction and quantification strategies for the downstream informatics. Through the data- driven development of rigorous probabilistic models for variant calling, we will achieve 1 in 1000 molecular detection sensitivity with high positive predictive value (PPV) for the clinical testing of liquid biopsy specimens. We propose the following two specific aims: Aim 1: Develop a new molecular barcoding method for liquid biopsy samples using a novel temperature- toggled hairpin looped primer and barcode. Aim 2: Improve the analysis of liquid biopsy samples using a robust bioinformatics analysis pipeline tailored to PCR-based molecular barcoded sequencing data. Ultimately, this proposal seeks to provide easy-to-use, high performance, and 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.
