Integrated system for preparation of NGS libraries from crude biological samples

描述(由申请人提供)：建议的SBIR计划的总体目标是开发一种可以接受全血作为输入样本的仪器系统，并产生适合于下一代DNA测序(NGS)的基因组DNA文库。所有步骤，从DNA提取到最终文库构建，都是通过一个单一的自动化过程进行的，无需任何用户干预。由于全自动化流程，所提出的系统将大大降低NGS测序的成本和人力，并加快NGS技术进入临床诊断环境。该仪器的另一个优点是，它应该可以从100‘S毫升的血液扩展到单细胞水平。在癌症研究和诊断中，将NGS技术应用于小样本是一个日益重要的目标。该系统采用了一种新的微流控样品制备方法。一种单一的连续流动技术被用来操纵和分离细胞、亚细胞细胞器和大的基因组DNA分子(表现出颗粒状的属性)。对所有样品制备步骤使用公共技术提供了几个好处：1)可以在单个消耗品设备上的单个操作中完成多个顺序处理步骤，从而简化了系统设计。2)在多步骤过程中，反应步骤和反应后清理步骤的集成实现了处理步骤之间样品的无缝、潜在的零损失转移。3)样品的纯化仅以“颗粒”大小为依据。不使用对固相的差示吸附，避免了由于不完全洗脱造成的样品损失。4)可以直接将大多数NGS平台中常见的冗长、复杂的样品制备协议自动化，从而使NGS技术更容易、更具成本效益地应用于临床试验研究和诊断测试等高通量应用中。 公共卫生相关性：拟议的SBIR计划的总体目标是开发一种可以接受全血作为输入样本的仪器系统，并产生适合于下一代DNA测序(NGS)的基因组DNA文库。所有步骤，从DNA提取到最终文库构建，都是通过一个单一的自动化过程进行的，无需任何用户干预。由于全自动化流程，所提出的系统将大大降低NGS测序的成本和人力，并加快NGS技术进入临床诊断环境。

DESCRIPTION (provided by applicant): The overall goal for the proposed SBIR program is to develop an instrument system that can accept whole blood as an input sample and produce a genomic DNA library suitable for next- generation DNA sequencing (NGS). All steps, from DNA extraction to final library construction, take place by a single automated process, without any user intervention. Because of the fully automated process, the proposed system would greatly lower the cost and labor of NGS sequencing, and accelerate movement of NGS technology into clinical diagnostic settings. Another advantage of the instrument is that it should be scalable from 100's of ml of blood down to the single cell level. Application of NGS technology to small samples is an increasingly important goal in cancer research and diagnostics. The proposed system utilizes a novel approach to microfluidic sample preparation. A single continuous-flow technology is used to manipulate and separate cells, subcellular organelles, and large genomic DNA molecules (that exhibit particle-like properties). The use of a common technology for all sample prep steps provides several benefits: 1) Multiple sequential processing steps can be accomplished in a single operation, on a single consumable device, thereby simplifying system design. 2) In multi-step processes, integration of reaction steps and post-reaction cleanup steps enables seamless, potentially zero-loss transfer of sample between processing steps. 3) Sample purification is accomplished on the basis of "particle" size alone. Differential adsorption to solid phases is not used, and sample loss due to incomplete elution is voided. 4) It is straightforward to automate the lengthy, complex sample preparation protocols that are typical of most NGS platforms, making it easier and more cost effective to move NGS technology into high throughput applications like clinical trials research and diagnostic testing. PUBLIC HEALTH RELEVANCE: The overall goal for the proposed SBIR program is to develop an instrument system that can accept whole blood as an input sample and produce a genomic DNA library suitable for next- generation DNA sequencing (NGS). All steps, from DNA extraction to final library construction, take place by a single automated process, without any user intervention. Because of the fully automated process, the proposed system would greatly lower the cost and labor of NGS sequencing, and accelerate movement of NGS technology into clinical diagnostic settings.