Multiplexed Assembly and Annotation of Synthetic Biology Constructs Using Long-Read Nanopore Sequencing.

Area of Science:

• Biotechnology and Synthetic Biology
• Molecular Biology and Genomics

Background:

• Plasmid DNA is crucial for biotechnology and medicine, serving as a vehicle for recombinant DNA.
• Traditional Sanger sequencing for plasmid validation is time-consuming and struggles with complex sequences.
• Advancements in DNA synthesis necessitate scalable validation methods for complex plasmids.

Purpose of the Study:

• To prototype a high-throughput plasmid sequencing method.
• To develop a scalable and cost-effective alternative to Sanger sequencing for plasmid validation.
• To enable comprehensive characterization of plasmid DNA, including epigenetic marks and contamination.

Main Methods:

• Utilized DNA transposition for library preparation.
• Employed Oxford Nanopore sequencing for long-read data generation.
• Developed a method named Circuit-seq for de novo plasmid assembly and analysis.

Main Results:

• Circuit-seq generated robust, full-length, and accurate plasmid assemblies irrespective of sequence complexity.
• The method produced contiguous plasmid maps for diverse plasmid sizes and constructs.
• Long-read data allowed for characterization of epigenetic modifications and estimation of plasmid contamination.

Conclusions:

• Circuit-seq provides a scalable, cost-effective, and rapid solution for plasmid validation.
• The technology accelerates key steps in synthetic biology research and development.
• Low equipment costs make Circuit-seq accessible for individual research laboratories.