Shapland Elaine B, Holmes Victor, Reeves Christopher D, Sorokin Elena, Durot Maxime, Platt Darren, Allen Christopher, Dean Jed, Serber Zach, Newman Jack, Chandran Sunil
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
‡TOTAL New Energies USA, Inc., 5858 Horton Street, Suite 253, Emeryville, California 94608, United States.
ACS Synth Biol. 2015 Jul 17;4(7):860-6. doi: 10.1021/sb500362n. Epub 2015 May 6.
In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.
近年来，新一代测序（NGS）技术大幅降低了全基因组测序的成本，而通过桑格测序法进行质粒序列验证的成本仍然很高。因此，工业规模的菌株工程师要么限制设计数量，要么在质量控制方面走捷径。在此，我们表明，在一次Illumina MiSeq运行中，可以以每个不到3美元的成本（15倍覆盖率）对4000多个质粒进行完整测序，这比使用桑格测序法（2倍覆盖率）降低了20倍。我们将Nextera转座反应的体积减少了100倍，并开发了一种自动化工作流程来制备数千个用于测序的样品。我们还开发了软件来跟踪样品和相关序列数据，并快速识别缺陷最少的正确组装构建体。随着DNA合成和组装成为一种集中供应的商品，这种NGS质量控制（QC）流程对于运行高通量DNA构建管道的团队至关重要。
