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

描述(申请人提供)：下一代测序的靶向浓缩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意义，并增加测序覆盖深度。这项STTR提案的目标是开发并商业化一种能够无扩增的百万碱基靶标序列浓缩的新技术。这项技术与目前市场上所有其他浓缩方法有很大的不同。我们的方法，MESA(兆数据库浓缩无扩增)，在微流控液滴中分离百万碱基大小的基因组DNA分子，并执行TaqMan聚合酶链式反应，以确认所需目标序列的存在。然后，在DNA测序之前，对TaqMan鉴定的液滴进行分类和收集，以丰富目标序列。这种方法是独一无二的，因为它是唯一一种分离完整的百万碱基大小的片段的方法，而且不需要DNA扩增。该方法中的TaqMan聚合酶链式反应仅用于鉴定目标序列，不用于通过扩增本身进行丰富的目的。此外，在测序之前，从浓缩的样品中去除TaqMan扩增片段。所有其他现有的靶向浓缩方法都将DNA片断，并通过聚合酶链式反应扩增或基于杂交的序列捕获进行浓缩。这两种方法都受到各种限制，包括缺乏一致性、特异性、覆盖深度或放大导致的偏见/伪影。此外，这些方法通常既昂贵又耗时，需要执行数千个探针或引物组。在这项提案中，我们的目标是使用一种工作流程来丰富人类基因组的一个巨型数据库区域，该工作流程产生比目前可能的更统一、更具体和更高覆盖率的序列数据。MESA方法的智力优势来自于它能够为靶向序列浓缩提供快速、更强大和低成本的解决方案。当与较新的、长时间的测序程序相结合时，MESA方法还有望帮助识别新的疾病相关突变，这些突变映射到基因组中难以测序的区域。MESA方法的商业化通常将提高靶序列浓缩的效用，并进一步使从事不同生物学领域的研究人员采用这种方法。

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.