Target Insertion Amplification and Sequence (TIAS): A novel targeted sequencing technology that performs rapid target enrichment and next generation sequencing sample preparation simultaneously

该提案的目标是建立一种快速、特异、灵敏和廉价的诊断靶点富集方法 应用于人类健康和流行病防范的技术，如病毒监测、微生物耐药性 检测以及体细胞肿瘤和液体活组织检查分析。第一阶段SBIR项目将创建一个新的诊断目标 富集技术，将执行快速目标富集和下一代测序（NGS）样品 同时制备，减少周转时间，并以最少的数据产生高度灵敏和特异的结果 藏物目前基于探针的NGS分析方法包括两个步骤：文库制备和靶点富集。 这些方法需要大量的DNA样品操作和在高温下孵育，以长时间地检测DNA。 这段时间会破坏DNA并在数据中引入伪影。这些伪影可能会混淆罕见变体的结果 检测异质性样品，如病毒准种、复杂微生物样品或循环肿瘤DNA。 虽然已经开发了采用独特分子标识符的方法，例如双链体测序和iDES， 为了解决这个问题，它们的应用有限，因为它们依赖于专门的生物信息学， 经鉴定确定的基因组特异性误差谱。RED Genomics正在开发靶向插入 扩增和测序（TIAS）探针，这是一种新型的基于探针的靶富集技术， 降低了成本和周转时间，同时提高了特异性和灵敏度，而不依赖于专门的生物信息学 或基因组误差分布。这是通过减少样品操作、孵育时间和温度来实现的， 最小化DNA损伤和数据伪影。TIAS探针具有独特的结构，允许捕获非- 在低温下变性双链DNA（或cDNA）以形成环状分子，其被扩增（通过PCR 或滚环扩增）以产生富含序列准备好的靶的NGS文库。传统的基于连接的NGS 文库制备可能是低效的并引入群体取样偏差。TIAS探头利用了 无缝克隆方案和原型数据表明它比连接更有效，因此TIAS探针增加了文库 转换率和减少整体错误。拟议研发的目标是使用 与基于标准探针的目标相比，TIAS探针具有更高的准确性和相当或更高的效率 富集方法这将通过测试几种蛋白质和化学试剂来实现， 稳定杂交后形成的中间分子。下一步，将制定成功的协议， 整合靶双链DNA和TIAS探针以产生共价闭合的环状双链DNA 分子。将进行靶向测序，并将质量指标数据与基于标准探针的 来自一个或多个商业供应商（即IntegratedDNATechnologies，TwistBioscience， Roche和Agilent）。TIAS技术将NGS样品制备和靶点富集相结合， 成本，节省时间，提供更准确的结果，为目前的目标浓缩套件提供了一个有竞争力的替代方案， 市场

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.