Tonight, I watched a recent Oxford Nanopore Technologies video about plasmid sequencing. The title is “A new and improved ‘gold standard’ for complete plasmid verification.” This session was a Knowledge Exchange with Aaron Pomerantz, Segment Marketing Manager at ONT. They spoke about plasmids and their use in molecular biology and industrial processes. Sanger sequencing has been used to confirm the accuracy of inserts. Nanopore sequencing does not require primers and can resolve repetitive sequences. Complicated primer walking is avoided. Whole plasmid verification has become accessible with Nanopore. A complete plasmid sequencing workflow is now available, enabling same-day turnaround. Jemma Jordan, the Director of Product Management at ONT, explained the wet lab workflow for plasmid sequencing. Jordan noted that sequencing with ONT can fit into current lab workflows. Up to ninety-six samples can be processed simultaneously with the Rapid Barcoding kit and MinION flow cells in two hours. After plasmid purification, plasmid DNA can be cleaved with the Rapid Barcoding transposase. Samples can be pooled and cleaned together. Jordan explained that the most cost-effective way of using this workflow is with 96 samples at once. Jordan emphasized that no third-party primers or reagents are needed. Stephen Rudd, Director of Bioinformatics Protocols at ONT, described the bioinformatics analysis of plasmid sequences. The recommended approach uses the wf-clone-validation workflow in EPI2ME. The workflow is packaged to promote reproducibility and functionality. The workflow takes a folder of multiplexed or demultiplexed sequences. The sequences are filtered by length to a reasonable range. Host sequences are removed, if wanted. The assembly is based on Tricycler and Flye. The single consensus sequence is prepared and polished with Medaka. Plannotate annotates the assembly. The cloned insert sequence is inferred based on primers. The workflow requires the sequence path and the assembled plasmids’ expected size. Several parameters can be modified. The report is generated as an HTML file. The number of reads observed and used for the assembly can be toggled. The plasmid map is displayed with features. The status and length of the assembled plasmid is displayed for each barcode. The length of the cloned insert observed is calculated depending on the cloning primers used. A folder of output files includes the consensus sequence and a bed file. A sample sheet allows the user to customize the expected sizes for each plasmid/barcode. I have used this workflow several times, and it ran in 20-30 minutes. Rudd did mention that additional customizations would become available.
