Suckling Lorna, McFarlane Ciaran, Sawyer Chelsea, Chambers Stephen P, Kitney Richard I, McClymont David W, Freemont Paul S
The London DNA Foundry, SynbiCITE, Imperial College London, London, SW7 2AZ, UK.
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
Synth Syst Biotechnol. 2019 Jan 22;4(1):57-66. doi: 10.1016/j.synbio.2019.01.002. eCollection 2019 Mar.
High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina Nextera XT technology and the Labcyte Echo acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.
用于下一代测序（NGS）的质粒DNA文库的高通量制备是分子生物学实验室的一项重要能力。特别是，当产生大量质粒变体时，这是一项必不可少的质量控制（QC）检查。在这里，我们描述了使用实验设计（DOE）方法，利用Illumina Nextera XT技术和Labcyte Echo声学液体分配系统，优化用于NGS的质粒DNA文库的小型化制备。此外，我们还描述了可作为QC检查实施的方法，用于鉴定质粒DNA样品中基因组DNA（gDNA）的存在以及随后对gDNA的剪切，否则gDNA会阻止质粒DNA的声学转移。这种工作流程能够制备出产生高质量测序数据的质粒DNA文库。
