Target insertion amplification and sequence (tias): a novel targeted sequencing technology that performs rapid target enrichment and next generation sequencing sample preparation simultaneously -…

Target insertion amplification and sequence (tias): a novel targeted sequencing technology that performs rapid target enrichment and next generation sequencing sample preparation simultaneously - the goal of this proposal is the creation of a rapid, specific, sensitive and inexpensive diagnostic target enrichment technology with applications in human health and pandemic preparedness such as viral surveillance, microbial resistance detection, and somatic tumor and liquid biopsy analysis. This phase i sbir project will create a novel diagnostic target enrichment technology that will perform rapid target enrichment and next generation sequencing (NGS) sample preparation simultaneously, reducing turn-around time and yielding highly sensitive and specific results with minimal data artifacts. Current methods for probe-based NGS analysis consist of two steps: library preparation and target enrichment. These methods require significant DNA sample manipulation and incubations at elevated temperatures for prolonged periods of time that damage DNA and introduce artifacts in the data. These artifacts can confound results for rare variant detection in heterogeneous samples such as viral quasispecies, complex microbial samples or circulating tumor DNA. While methods that employ unique molecular identifiers, such as duplex sequencing and ides, have been developed to address this issue, they have limited application due to their reliance on specialized bioinformatics that require empirically-determined error profiles that are gene panel specific. Red genomics is developing target insertion amplification and sequence (tias) probes, which is a novel probe-based target enrichment technology that dramatically reduces cost and turnaround time while increasing specificity and sensitivity without relying on specialized bioinformatics or gene panel error profiles. This is achieved by decreasing sample manipulation, incubation times and temperature, which minimizes DNA damage and data artifacts. Tias probes have a unique structure that allows the capture of non- denatured double stranded DNA (or cdna) at low temperature to form a circular molecule which is amplified (via PCR or rolling circle amplification) to yield a sequence-ready target enriched NGS library. Traditional ligation-based NGS library preparation can be inefficient and introduce population sampling bias. Tias probes leverage methods used in seamless cloning protocols and prototype data shows IT to be more efficient than ligation, so tias probes increase library conversion rates and reduce overall errors. The goal of the proposed r & d is to demonstrate target enrichment using tias probes with increased accuracy and comparable or better efficiency compared to standard probe-based target enrichment methods. This will be accomplished by testing several protein and chemical reagents that enhance and stabilize the intermediate molecule formed after hybridization. Next, protocols will be developed for the successful integration of the target double stranded DNA and tias probes to yield covalently closed circular double stranded DNA molecules. Targeted sequencing will be performed and quality metric data will be compared to standard probe-based target enrichment protocols from one or more commercial vendors (i.e. integrated DNA technologies, twist bioscience, roche and agilent). By combining NGS sample preparation and target enrichment, tias technology will reduce reagent costs, save time and deliver more accurate results, offering a competitive alternative to current target enrichment kits on the market.