Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process

详细信息    查看全文

• 作者：Elaine B. Shapland ; Victor Holmes ; Christopher D. Reeves ; Elena Sorokin ; Maxime Durot ; Darren Platt ; Christopher Allen ; Jed Dean ; Zach Serber ; Jack Newman ; Sunil Chandran
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：July 17, 2015
• 年：2015
• 卷：4
• 期：7
• 页码：860-866
• 全文大小：356K
• ISSN：2161-5063

文摘

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脳 coverage), which is a 20-fold reduction over using Sanger sequencing (2脳 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.