Laboratory for MEMS Applications, Department of Microsystems Engineering IMTEK, University of Freiburg, Germany.
Molecular and Experimental Mycobacteriology, Forschungszentrum Borstel, Borstel, Germany; German Center for Infection Research, Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany.
Biotechnol Adv. 2020 Jul-Aug;41:107537. doi: 10.1016/j.biotechadv.2020.107537. Epub 2020 Mar 19.
Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.
下一代测序技术正在从一种用于研究目的的技术演变为一种应用于临床诊断的技术。最近推出的高通量和台式仪器提供了更低的每个碱基成本和更快的检测时间的全自动测序运行。反过来，复杂而繁琐的文库制备，从分离的核酸开始，最终得到带有测序接头的扩增和条形码 DNA，已被确定为一个重大瓶颈。文库制备方案通常由多步过程组成，需要昂贵的试剂和大量的手工时间。需要相当重视标准化，以确保稳健性和可重复性。本文综述了下一代测序文库制备自动化的现状。概述了与文库制备相关的主要挑战，并根据其功能原理对不同的自动化策略进行了分类。移液工作站允许高通量处理，但提供的灵活性有限，而微流控解决方案由于微型化和降低投资成本而具有巨大的潜力。例如，对于新兴的单细胞转录组学领域，微流控技术可将数万细胞单个化为纳升液滴，并对 RNA 进行条形码标记，以将每个核酸序列分配给其原始细胞。最后，概述了两个应用，细菌病原体的特征和人类免疫遗传学中的测序，并讨论了自动化的好处。
