Multiplexed long-read plasmid validation and analysis using OnRamp.

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Recombinant plasmid vectors are crucial in various biological fields.
• Current plasmid validation methods like Sanger sequencing have limitations in read length, scalability, and sequencing through secondary structures.
• High-throughput sequencing is effective for large-scale validation but impractical for routine use.

Purpose of the Study:

• To introduce OnRamp, a novel method for routine plasmid validation using Oxford Nanopore sequencing.
• To provide a cost-effective and scalable alternative to Sanger sequencing for full-plasmid analysis.
• To enable accessible long-read sequencing for plasmid validation, regardless of user programming expertise.

Main Methods:

• Development of customized wet-laboratory protocols for plasmid preparation.
• Creation of a bioinformatics pipeline for analyzing nanopore sequencing data.
• Deployment of the analysis pipeline on the OnRamp web application for user-friendly access.

Main Results:

• OnRamp successfully obtains full sequences from pooled plasmids.
• The method accurately detects sequence variations, even in regions with high secondary structure.
• OnRamp provides full-plasmid coverage and scalability at less than half the cost of Sanger sequencing.

Conclusions:

• OnRamp is a viable and accessible alternative for routine plasmid validation.
• Leveraging nanopore's long-read technology, OnRamp overcomes the limitations of existing methods.
• This approach facilitates wider adoption of long-read sequencing for plasmid analysis in research and diagnostics.