Microbead INtegrated DNA Sequencer (MINDS) System

描述（由申请人提供）：该合作项目旨在开发一种“微珠整合DNA测序仪”（MINDS），该测序仪有效地整合了DNA测序中的所有主要步骤，从文库构建到利用低成本微流体装置的最终序列输出。自动化MINDS系统将结合联合收割机三个基本步骤：1）使用微珠集落技术的文库构建、扩增和选择，2）纳升循环测序样品制备和纯化，以及3）DNA测序片段的基于微加工毛细管阵列电泳（uCAE）的分离。文库构建和扩增过程将输入剪切的、大小确定的DNA片段，并在珠上构建模板的乳液PCR扩增文库，每个珠代表单个DNA片段。然后在25 nL循环测序反应器中处理单珠以产生荧光标记的测序片段，所述测序片段使用芯片上亲和捕获被有效捕获、浓缩和纯化。然后在经验证的微制造uCAE测序仪上分离片段并确定大小。 这项提案将联合收割机的努力，微芯片生物技术公司。(MBI)与三个合作学术机构签订了分包合同。MBI将开发一个基于微芯片的DNA样品制备纳米级热循环模块原型和一个先进的旋转扫描仪，该扫描仪具有使用常规外部化学的原型uCAE测序系统。然后将其整合以产生具有25 nL循环测序样品制备、亲和纯化和uCAE测序的阵列的MINDS微芯片。在30个月内完成这项工作后，MBI将进一步整合Mathies实验室正在开发的基于微珠的库技术，以创建400个通道的MINDS系统原型，为测试做好准备。这些发展将建立在学术合作者共同开发的新方法和策略的基础上，特别是uCAE分离系统和基于微珠的微流体“克隆”方法。加州大学数学实验室的一位教授。伯克利分校将支持新的微技术的发展，用于克隆的扩增和选择，以及这些方法和过程与原型微制造测序系统的集成。西北大学的巴伦实验室与Mathies合作，将开发和测试新型DNA分离基质，这些基质很容易加载到芯片微通道中并从芯片微通道中替换，并提供快速，高分辨率的分离，至少有700个碱基读取。对哥伦比亚基因组中心Ju实验室的支持将支持基因组克隆生产新方法的开发，以及MBI生产的集成测序系统的β测试。哥伦比亚研究小组还将与伯克利研究小组合作，改进克隆生产和选择的方法，以及芯片上样品的清理。该项目的目标是在哥伦比亚基因组中心放置一个完全集成的原型桑格测序系统的测试版，并证明其以低100倍的成本进行基因组测序和重测序的能力，以及通过产生超过1.5千兆碱基的鸟枪序列来实现约700万碱基/天/机器的通量。 MINDS系统将大大降低鸟枪测序和重测序的成本，通过利用完善的uCAE设备的能力，通过制备更接近分析要求的样品体积来分析亚纳升体积，将循环测序试剂消耗减少100倍。文库构建将以基于珠的形式自动化，扩增和选择在单个批量反应中以全规模进行，再次降低试剂消耗和成本。为微芯片设计的新型聚合物分离基质已经显示出良好的性能，并且沿着微流体体积的减小，将使基质费用最小化。有了这些组合的创新，MINDS系统将推动CAE仪器接近四色桑格荧光DNA测序的最终性能，在超高通量的实施基因组中心。未来的工作将探索适合于核心实验室和单个实验室的低通量版本的开发。

DESCRIPTION (provided by applicant): This collaborative project is aimed at the development of a "Microbead INtegrated DNA Sequencer" (MINDS) that efficiently integrates all of the major steps in DNA sequencing, from library construction to final sequence output exploiting low-cost microfluidic devices. The automated MINDS system will combine three fundamental steps: 1) library construction, amplification, and selection using microbead colony technologies 2) nanoliter cycle sequencing sample preparation and purification, and 3) microfabricated capillary array electrophoresis (uCAE)-based separation of DNA sequencing fragments. The library construction and amplification process will input sheared, sized DNA fragments and construct an emulsion PCR amplified library of template on beads, with each bead representing a single DNA fragment. Single beads will then be processed in a 25 nL cycle sequencing reactor to produce fluorescently labeled sequencing fragments that are efficiently captured concentrated and purified using on-chip affinity capture. The fragments are then separated and sized on a proven microfabricated uCAE sequencer. This proposal will combine the efforts of Microchip Biotechnologies Inc. (MBI) with subcontracts to three collaborating academic institutions. MBI will develop a prototype microchip-based DNA sample preparation nanoscale thermal cycling module and an advanced rotary scanner with a prototype uCAE sequencing system using conventional external chemistries. These will then be integrated to produce a MINDS microchip with arrays of 25 nL cycle sequencing sample preparation, affinity purification, and uCAE sequencing. When this has been accomplished, by 30 months, MBI will further integrate the microbead-based library technology being developed by the Mathies laboratory to create 400 channel MINDS System prototypes ready for beta-testing. These developments will build upon novel methods and strategies developed in tandem by the academic collaborators, in particular the uCAE separation system and bead-based microfluidic "cloning" methods. A subcontract to the Mathies lab at U.C. Berkeley will support the development of new microtechnologies for the amplification and selection of clones, and the integration of these methods and processes with prototype microfabricated sequencing systems. In collaboration with Mathies, the Barron lab at Northwestern will develop and test novel DNA separation matrices that are easily loaded into and replaced from chip microchannels, and that provide rapid, high-resolution separations with at least a 700-base read. A subcontract to the Ju lab at the Columbia Genome Center will support the development of new methods for genomic clone production as well as for beta-testing the integrated sequencing systems produced by MBI. The Columbia group will also work with the Berkeley group toward improving methods for clone production and selection, and for on-chip sample clean-up. The project goal is to place a beta version of the fully integrated, prototype Sanger sequencing system at Columbia Genome Center and to demonstrate its capability to perform genomic sequencing and resequencing at 100-fold lower cost and a throughput of about 7 million bases/day/machine by producing over 1.5 gigabase of shotgun sequence. The MINDS system will greatly reduce the cost of shotgun sequencing and resequencing, by exploiting the ability of well established uCAE devices to analyze sub-nanoliter volumes through preparation of samples in volumes more closely matched to the analytical requirements, reducing cycle sequencing reagent consumption by 100-fold. Library construction will be automated in the bead-based format, with amplification and selection performed at full scale in a single bulk reaction, again reducing reagent consumption and cost. A novel polymeric separation matrix designed for microchips already shows good performance and, along with microfluidic volume reductions, will minimize matrix expense. With these combined innovations, the MINDS system will drive CAE instrumentation close to the ultimate performance possible for four-color Sanger fluorescent DNA sequencing in an ultra-high-throughput implementation for genome centers. Future work will explore the development of lower-throughput versions appropriate for core and individual laboratories.