Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.
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.
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Elaine B
EB
Shapland
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Victor
V
Holmes
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Christopher D
CD
Reeves
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Elena
E
Sorokin
†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.
Maxime
M
Durot
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Darren
D
Platt
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Christopher
C
Allen
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Jed
J
Dean
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Zach
Z
Serber
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Jack
J
Newman
†Amyris, Inc., 5885 Hollis, Suite 100, Emeryville, California 94608, United States.
Sunil
S
Chandran
D004247:DNA / Q000032:analysis* / Q000378:metabolism; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods* / Q000592:standards; D054643:INDEL Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods* / Q000592:standards
D004247:DNA
NGS; high throughput; next-generation sequencing; synthetic biology