Amplification-free megabase target-enrichment for next-generation sequencing

用于下一代测序的无扩增兆碱基靶标富集

• 批准号: 8782103
• 负责人: Dennis J Eastburn
• 金额: $ 21.5万
• 依托单位: MISSION BIO, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-04 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782103
• 关键词: Adoption; Area; Base Sequence; Benchmarking; Biological Assay; Biology; DNA; DNA Sequence; DNA amplification; Data; Data Analyses; Detection; Disease; Drops; Genetic; Genetic Research; Genome; Genomic DNA; Genomics; Goals; Human Genome; Investigation; Length; Maps; Marketing; Methods; Microfluidics; Mutation; Organism; Phase; Procedures; Reaction; Reading; Recovery; Research Personnel; Sampling; Sensitivity and Specificity; Sequence Analysis; Side; Small Business Technology Transfer Research; Solutions; Sorting - Cell Movement; Specificity; Technology; Time; Vision; Work; base; commercialization; cost; cost effective; digital; genome sequencing; human disease; instrument; instrumentation; interest; new technology; next generation sequencing; novel; novel strategies; prototype; public health relevance; stem; tool; user-friendly

DESCRIPTION (provided by applicant): Target enrichment for next-generation sequencing enables researchers to reduce the burden of sequence data analysis, obtain higher levels of statistical significance by examining more genomes and increase sequencing coverage depth. The goal of this STTR proposal is to develop and commercialize a novel technology capable of amplification-free megabase target sequence enrichment. This technology is highly differentiated from all other enrichment approaches currently on the market. Our method, MESA (Megabase Enrichment Sans Amplification), isolates large megabase-sized genomic DNA molecules in microfluidic drops and performs TaqMan PCR reactions to confirm the presence of the desired target sequence. The TaqMan identified drops are then sorted and collected to enrich for the target sequence prior to DNA sequencing. This approach is unique in that it is the only method that isolates intact megabase-sized fragments and does so without amplification of the DNA. The TaqMan PCR reaction in this procedure is used only to identify target sequences and not for the purpose of enriching through amplification itself. Furthermore, the TaqMan amplicons are removed from the enriched sample prior to sequencing. All other existing methods for target- enrichment fragment the DNA and enrich through PCR amplification or hybridization-based sequence capture. Both approaches suffer from various limitations including, lack of uniformity, specificity, depth of coverage or amplification induced bias/artifacs. Additionally, these approaches are often costly and time consuming, requiring the implementation of thousands of probes or primer sets. In this proposal, we aim to demonstrate enrichment of a megabase region of the human genome using a workflow that generates more uniform, specific and high coverage sequence data than is currently possible. The intellectual merits of the MESA approach stem from its ability to deliver a rapid, more robust and low-cost solution for targeted sequence enrichment. When combined with newer, long-read sequencing procedures, the MESA method also promises to aid in identifying new disease associated mutations that map to difficult to sequence regions of the genome. Commercialization of the MESA approach will generally enhance the utility of target sequence enrichment and further its adoption by researchers working in diverse areas of biology.

描述（由申请人提供）：下一代测序的靶标富集使研究人员能够减轻序列数据分析的负担，通过检查更多的基因组获得更高水平的统计显著性，增加测序覆盖深度。这项STTR提案的目标是开发和商业化一种能够无扩增的百万碱基靶序列富集的新技术。该技术与目前市场上所有其他富集方法高度不同。我们的方法，MESA (Megabase Enrichment Sans Amplification)，在微流控液滴中分离出大的Megabase大小的基因组DNA分子，并进行TaqMan PCR反应以确认所需目标序列的存在。然后对TaqMan鉴定的液滴进行分类和收集，以便在DNA测序之前为目标序列进行富集。这种方法的独特之处在于，它是唯一一种分离完整的兆酶大小片段的方法，而且不需要扩增DNA。本程序中的TaqMan PCR反应仅用于鉴定目标序列，不用于通过扩增本身进行富集。此外，测序前从富集样品中去除TaqMan扩增子。所有其他现有的目标富集方法都是通过PCR扩增或基于杂交的序列捕获来对DNA进行片段化和富集。这两种方法都有各种局限性，包括缺乏一致性、特异性、覆盖深度或放大引起的偏差/伪影。此外，这些方法通常是昂贵和耗时的，需要实现数千个探针或引物集。在本提案中，我们的目标是证明人类基因组的一个大碱基区域的富集使用工作流，生成比目前可能的更统一，特异性和高覆盖率的序列数据。MESA方法的优势在于它能够为目标序列富集提供快速、更强大和低成本的解决方案。当与较新的长读序列测序方法相结合时，MESA方法还有望帮助识别新的疾病相关突变，这些突变映射到难以测序的基因组区域。MESA方法的商业化通常会提高目标序列富集的效用，并进一步被生物学不同领域的研究人员采用。