Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies' MinION device

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Plasmid sequence verification is critical for reagents and therapeutics.
• Capillary-based Sanger sequencing is standard but costly, especially in regulated environments.
• High-throughput, accurate plasmid sequencing is needed for clinical research.

Purpose of the Study:

• To develop a cost-effective and accurate plasmid sequencing and consensus generation procedure.
• To offer an alternative to expensive capillary-based sequencing for plasmid verification.
• To enable accessible, high-quality plasmid sequencing for clinical applications.

Main Methods:

• Utilized Oxford Nanopore Technologies' MinION device for plasmid sequencing.
• Employed a full MinION flow cell per plasmid for maximized data and stringent quality filtering.
• Implemented pseudopairing reads and a pileup consensus approach for reduced error rates and confidence scoring.

Main Results:

• Achieved a significant reduction in read error rates from 5.3% to 0.53% through pseudopairing.
• Demonstrated 100% accuracy in consensus sequences for pure plasmid samples.
• Showcased sensitivity in detecting small mutations (insertions, deletions, SNVs) and subclonal variants.

Conclusions:

• The developed pipeline offers substantial cost savings for clinical-grade plasmid sequencing.
• Provides accessible, high-quality plasmid sequence verification for clinical research.
• Delivers complete and accurate sequence verification suitable for applications requiring absolute accuracy.