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

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Sanger sequencing remains costly for plasmid verification, hindering industrial-scale strain engineering and quality control.
• High-throughput DNA construction pipelines require efficient and cost-effective quality control methods for plasmids.

Purpose of the Study:

• To develop a cost-effective next-generation sequencing (NGS) workflow for high-throughput plasmid verification.
• To reduce the cost per plasmid sequenced compared to traditional Sanger sequencing.

Main Methods:

• Utilized Illumina MiSeq for sequencing over 4000 plasmids with 15× coverage.
• Reduced Nextera tagmentation reaction volume by 100-fold and implemented an automated sample preparation workflow.
• Developed custom software for sample tracking, data management, and defect identification in assembled constructs.

Main Results:

• Achieved a cost of less than $3 per plasmid, a 20-fold reduction compared to Sanger sequencing.
• Demonstrated the feasibility of sequencing thousands of plasmids in a single NGS run.
• Successfully identified correctly assembled constructs with minimal defects using the developed software.

Conclusions:

• NGS offers a significantly more economical and efficient approach for plasmid quality control in high-throughput pipelines.
• The developed automated NGS workflow and software are crucial for advancing DNA construction and strain engineering.
• This method addresses the limitations of Sanger sequencing, enabling broader adoption of comprehensive quality control in synthetic biology.