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

The present study describes a workflow for nanopore sequencing designed to maximize plasmid DNA yield and sequencing accuracy. Researchers cover each step of the process, which includes plasmid DNA extraction, library preparation with rapid barcoding and adapter ligation, loading of the library onto a flow cell for sequencing, base calling (in real time or post-hoc), and sequence assembly using Autocycler. The authors also specify the computer requirements, which largely depend on whether basecalling is done in real time. The authors sequenced plasmids purified from ten clinical strains using this workflow. To remove host-cell strain differences as a variable, the authors  also conjugated these clinical strains with a common recipient strain and sequenced the transconjugants. These experiments produced plasmid assemblies that match plasmid sizes inferred from gel mobility and that, in 2/3 of cases, also match short-read polished assemblies or are within 0.05% pairwise sequence identity. The enrichment of plasmid DNA sequences also maximizes the efficiency of plasmid sequencing, allowing parallel sequencing of up to 24 samples with optimal coverage. This protocol is adequate for plasmids ranging from 4 to 174 kb and tolerates up to 72% chromosomal contamination without affecting accuracy. This protocol is ideal for situations where the chromosomal sequence is already known or of no interest, such as for plasmid sequence verification, improving the accuracy of plasmid sequences already obtained by WGS, or sequencing plasmids captured by conjugation. The organism used to illustrate this work is Escherichia coli, which is representative of hosts used for recombinant gene expression and for plasmid capture through conjugation.