Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore.
PLoS One. 2013 Jul 24;8(7):e64084. doi: 10.1371/journal.pone.0064084. Print 2013.
Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.
用于下一代 DNA 测序（NGS）的文库制备仍然是测序过程中的一个关键瓶颈，可以通过改进自动化和小型化来缓解。我们描述了一种用于自动化实验室方案的微流控设备，该方案需要一个或多个柱层析步骤，并展示了其在为 Illumina 和 Ion Torrent 平台制备下一代测序文库中的实用性。与手动文库制备相比，该设备可以同时生成 16 种不同的文库，同时显著降低试剂成本和人工操作时间。使用适当的柱基质和缓冲液，可以在末端修复、dA 加尾和接头连接后在芯片上进行大小选择，因此从芯片上洗脱的文库可以直接用于测序。该设备的核心架构确保了均匀、可重复的柱包装，无需用户监督，并适应任何顺序的多个常规方案步骤，例如试剂混合和孵育；柱包装、加载、洗涤、洗脱和再生；洗脱产物的捕获，用作方案中后续步骤的底物；以及去除一个柱基质，以便在同一方案中使用具有不同功能特性的两个或更多个柱基质。微流控设备安装在塑料载体上，因此可以使用标准移液器和液体处理机器人进行试剂和产品的分配和回收。装有载体的设备使用台式控制器进行操作，该控制器可密封并通过可编程温度控制来操作设备，从而消除了用户手动连接管或连接器的任何要求。除了 NGS 文库制备外，该设备和控制器还适用于自动化其他需要柱层析步骤的耗时且容易出错的实验室方案，例如染色质免疫沉淀。
