Center for Biomedical Engineering, School of Engineering, Brown University, Providence, RI, USA.
Sci Rep. 2024 May 31;14(1):12564. doi: 10.1038/s41598-024-63014-x.
Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption-desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations.
下一代测序(NGS)正在成为一种强大的分子诊断工具,但仍受到繁琐且低效的样本制备的限制。我们提出了一种新颖的自动化 NGS 文库制备系统,该系统具有简化的机械设计,利用宏观和微观流体特性来优化传热、反应动力学、传质、流体力学、吸附-解吸速率和分子热力学。我们的方法引入了一种独特的双套管圆柱形毛细管系统,该系统与可编程注射器泵和 Peltier 加热元件相连,能够高效地执行所有步骤。自动试剂移动、混合以及基于磁珠的洗涤与基于毛细管的热循环(毛细管-PCR)完全集成到一个单一平台中。通过优化预涂板,手动 3 小时的文库制备过程可减少至不到 15 分钟的实际操作时间,随后仪器运行时间不到 6 小时,在此期间无需用户交互。我们将该方法应用于两种具有不同 DNA 片段化要求的文库制备测定法(机械片段化与酶片段化),充分限制了每个运行的耗材使用量,每个运行仅需一个试剂盒和一个 384 孔板。我们的平台成功地并行制备了 8 个文库,生成了人源和大肠杆菌 DNA 文库的测序数据,与阳性对照相比,覆盖率偏差可忽略不计。我们文库的所有测序数据均达到 Phred(Q)分数>30,在 99%置信度下映射到参考基因组。该方法实现了与传统手动方法相当的最终文库浓度和大小分布,证明了其与下游测序和后续数据分析的兼容性。我们的工程设计在可测序文库的质量方面提供了重复性和一致性,强调了机械设计考虑因素的重要性,这些因素利用并优化了基本的流体力学和传热特性。此外,在这项工作中,我们提供了有关与自动化适应相比,NGS 文库制备测定中样品损失机制的独特见解,并指出了热力学、流体力学和传热原理可以改进未来机械设计迭代的领域。
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