Long-Read Plasmid Sequencing and Assembly Using Nanopore Sequencing-Based Workflows

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Plasmid DNA sequencing is crucial for various applications, including genetic engineering and pathogen surveillance.
• Existing methods can be limited by DNA yield, accuracy, and efficiency, especially with complex samples.

Purpose of the Study:

• To develop and validate a comprehensive nanopore sequencing workflow for maximizing plasmid DNA yield and accuracy.
• To provide detailed protocols for each step, from DNA extraction to sequence assembly.
• To assess the workflow's performance across different plasmid sizes and contamination levels.

Main Methods:

• Plasmid DNA extraction and purification.
• Library preparation using rapid barcoding and adapter ligation.
• Nanopore sequencing on flow cells.
• Real-time or post-hoc base calling and sequence assembly using Autocycler.
• Conjugation experiments to control for host strain variability.

Main Results:

• The workflow successfully produced accurate plasmid assemblies, matching gel-based size estimations and closely aligning with short-read polished assemblies.
• Achieved high accuracy (within 0.05% pairwise sequence identity) for 2/3 of sequenced plasmids.
• Demonstrated robustness for plasmids ranging from 4 to 174 kb, tolerating up to 72% chromosomal contamination.
• Enabled parallel sequencing of up to 24 samples with optimal coverage.

Conclusions:

• The described nanopore sequencing workflow is efficient and accurate for plasmid DNA analysis.
• It is suitable for plasmid sequence verification, improving whole-genome sequencing (WGS) data, and sequencing plasmids captured via conjugation.
• The protocol is adaptable for organisms like Escherichia coli, commonly used in recombinant gene expression and plasmid capture.