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

靶标插入扩增和测序（TIAS）：一种新颖的靶向测序技术，可同时进行快速靶标富集和下一代测序样品制备

• 批准号: 10758909
• 负责人: Phillip Gray
• 金额: $ 29.53万
• 依托单位: RED GENOMICS, INC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10758909
• 关键词: Address; Bacteriophage T4; Binding Proteins; Bioinformatics; Biological Sciences; Chemicals; Cloning; Complementary DNA; Complex; DNA; DNA Damage; DNA Integration; DNA Ligases; DNA-Directed DNA Polymerase; Data; Deoxyribonucleases; Detection; Diagnostic; Dideoxy Chain Termination DNA Sequencing; Digestion; Dropout; Escherichia coli; Escherichia coli Proteins; Exclusion; Exonuclease; Generations; Genomic DNA; Genomics; Goals; Health; Human; Incubated; Invaded; Libraries; Ligation; Marketing; Methods; Molecular; Molecular Weight; Morphologic artifacts; Outcome; Patients; Phase; Polyethylene Glycols; Population; Preparation; Property; Proteins; Protocols documentation; Reaction; Reagent; Reporting; Research; Resistance; Sampling; Sampling Biases; Sensitivity and Specificity; Single-Stranded DNA; Small Business Innovation Research Grant; Structure; System; Technology; Temperature; Testing; Time; Vendor; Viral; Virus; Work; base; cold temperature; cost; ds-DNA; gene panel; human pathogen; liquid biopsy; microbial; next generation sequencing; novel; novel diagnostics; nuclease; pandemic preparedness; prevent; prototype; rare variant; recombinase; research and development; respiratory virus; success; targeted sequencing; triple helix; tumor; tumor DNA; variant detection

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.

这项建议的目标是创建一种快速、特异、灵敏和廉价的诊断靶标浓缩 技术在人类健康和大流行预防中的应用，如病毒监测、微生物耐药性 检测，以及体瘤和液体活检分析。该第一阶段SBIR项目将创建一个新的诊断目标 将执行快速目标浓缩和下一代测序(NGS)样本的浓缩技术 同时准备，减少周转时间，以最少的数据产生高度敏感和特定的结果 手工艺品。目前基于探针的NGS分析方法包括两个步骤：文库制备和靶标富集法。 这些方法需要大量的DNA样本操作和在高温下长时间孵化 破坏DNA并在数据中引入伪像的时间段。这些人工产物可能会混淆稀有变体的结果 检测异质样本，如病毒准种、复杂微生物样本或循环中的肿瘤DNA。 而使用独特的分子识别符的方法，例如双链测序和IDE，已经发展到 为了解决这个问题，它们的应用有限，因为它们依赖于需要 经验性地确定的错误图谱是基因小组特有的。红色基因组公司正在开发靶标插入 扩增和测序(TIAS)探针是一种新的基于探针的靶浓缩技术，它显著地 降低成本和周转时间，同时提高特异性和敏感度，而无需依赖专门的生物信息学 或基因小组的错误特征。这是通过减少样品处理、孵化时间和温度来实现的， 从而将DNA损伤和数据伪装降至最低。TIA探测器具有独特的结构，允许捕获非 在低温下变性的双链DNA(或cDNA)形成环状分子，然后(通过聚合酶链式反应)进行扩增 或滚圈扩增)以产生序列就绪的靶标丰富的NGS文库。传统的基于连接的NGS 文库准备可能效率低下，并引入总体抽样偏差。TIA探测器利用 无缝克隆协议和原型数据表明，它比连接效率更高，因此TIAS探针增加了文库 提高了转换率，减少了总体误差。拟议的研发的目标是展示目标浓缩使用 与基于标准探头的靶标相比，TIA探头具有更高的精确度和相当或更高的效率 浓缩方法。这将通过测试几种蛋白质和化学试剂来实现，这些试剂可以增强和 稳定杂交后形成的中间分子。接下来，将为成功的 靶向双链DNA与TIAS探针的整合制备共价闭合环状双链DNA 分子。将执行定向测序，并将质量指标数据与基于标准探头的数据进行比较 来自一个或多个商业供应商的目标浓缩协议(即集成DNA技术、Twist Bioscience、 罗氏和安捷伦)。通过将NGS样品制备和靶向富集化相结合，TIAS技术将减少试剂 成本，节省时间并提供更准确的结果，为当前的目标浓缩试剂盒提供了一个具有竞争力的替代方案 市场。