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

描述（由申请人提供）：用于下一代测序的目标富集使研究人员能够减轻序列数据分析的负担，通过检查更多基因组获得更高水平的统计学显著性，并增加测序覆盖深度。该STTR提案的目标是开发和商业化一种能够进行无扩增meglobulin靶序列富集的新技术。这一技术与目前市场上所有其他浓缩方法有很大区别。我们的方法梅萨（Megabase Enrichment Sans Amplification）在微流体液滴中分离大的兆碱基大小的基因组DNA分子，并进行TaqMan PCR反应以确认所需靶序列的存在。然后将TaqMan鉴定的液滴分选并收集以在DNA测序之前富集靶序列。这种方法是独特的，因为它是唯一的方法，分离完整的兆碱基大小的片段，这样做没有扩增的DNA。本程序中的TaqMan PCR反应仅用于鉴定靶序列，而不是通过扩增本身进行富集。此外，在测序之前从富集的样品中去除TaqMan扩增子。用于靶富集的所有其他现有方法使DNA片段化并通过PCR扩增或基于杂交的序列捕获来富集。这两种方法都受到各种限制，包括缺乏均匀性、特异性、覆盖深度或扩增诱导的偏差/伪影。此外，这些方法通常是昂贵和耗时的，需要实施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证明人类基因组中一个巨大区域的富集，该工作流程可以产生比目前可能的更均匀，更特异和更高覆盖率的序列数据。梅萨方法的智力优势源于其能够为靶向序列富集提供快速、更稳健和低成本的解决方案。当与较新的长读序测序程序相结合时，梅萨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。梅萨方法的商业化通常将增强靶序列富集的效用，并进一步被生物学不同领域的研究人员采用。

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.