Sequencing complete plasmids on Oxford Nanopore Technologies sequencers using R2C2 and Chopper

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Plasmids are essential tools in molecular biology for various experimental applications.
• Accurate plasmid sequencing is crucial for verifying functionality, including correct insert and backbone.
• Traditional Sanger sequencing often only covered plasmid inserts, limiting full characterization.

Purpose of the Study:

• To adapt Oxford Nanopore Technologies R2C2 sequencing for rapid, cost-effective, full-length plasmid sequencing.
• To develop a bioinformatics pipeline (Chopper) for analyzing complete plasmid sequences.
• To provide an efficient method for plasmid quality control in research settings.

Main Methods:

• Adaptation of the R2C2 sequencing method from Oxford Nanopore Technologies.
• Development of the Chopper analysis pipeline for processing sequencing data.
• Testing the workflow using commonly used plasmids obtained from Addgene.

Main Results:

• Successfully generated highly accurate, full-length sequences of complete plasmids.
• Demonstrated the method's effectiveness for both individual and pooled plasmid sequencing runs.
• Achieved rapid and low-cost sequencing of plasmids.

Conclusions:

• The adapted R2C2 sequencing method and Chopper pipeline offer an efficient solution for complete plasmid sequencing.
• This workflow facilitates accurate plasmid verification, supporting molecular biology research.
• The method provides a valuable alternative to existing sequencing services, balancing throughput and cost.