Application Note
An in-house solution for whole plasmid characterization
Discover an efficient, end-to-end workflow for sequencing full-length plasmids directly in the lab.
Plasmids are the backbone of molecular biology, enabling advancements in gene therapy, genetic engineering, vaccine development, and more. Any errors in plasmid sequences can compromise experimental outcomes. By implementing an in-house sequencing workflow, researchers can achieve rapid, complete plasmid characterization without relying on external services, minimizing delays and enhancing data accuracy.
Download this application note to learn:
- A rapid, end-to-end workflow for complete, high-quality whole-plasmid characterization
- Best practices for library preparation and multiplexing for cost-effective analysis
- Advanced data analysis techniques for complete plasmid assembly and annotation
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iStock.com/Pogonici
Workflow overview | Whole-plasmid sequencing Rapid and complete plasmid characterisation with Oxford Nanopore sequencing Plasmids are the backbone of molecular biology, playing a pivotal role in applications such as gene therapy and vaccine development, genetic engineering, industrial biotechnology, and basic scientific research. Therefore, routine verification of plasmid constructs is an essential quality control step. Any errors in plasmids, such as mutations, truncations, or rearrangements, can compromise downstream experimental success. Oxford Nanopore sequencing enables the highly accurate, flexible, and secure characterisation of full-length plasmid sequences in-house without the need for primers, with results obtained in hours — negating the need to send constructs to third parties for validation. By obtaining full sequence data in a single experiment, the need for multiple techniques to confirm the identity of your constructs is also no longer required. Here we present a rapid, end-to-end workflow for complete, high-quality whole-plasmid characterisation using a MinION™ or GridION™ sequencing device and the EPI2ME™ analysis platform. Extraction: obtaining high molecular-weight DNA Library preparation: multiplexing samples Selecting an extraction method that will effectively remove contaminants — such as detergents, denaturants, chelating agents, or high salt concentrations — will ensure clean, high-quality DNA samples are taken forward to library preparation. We recommend the use of a plasmid midi prep kit, such as the QIAGEN Plasmid Plus Midi Kit, which enables extraction of reliable quantities of high-purity DNA from overnight cultures. For each sample, 50 ng of plasmid DNA is then taken forward into library preparation. We recommend the Qubit fluorometer for accurate DNA quantification. To prepare your library for sequencing and downstream analysis, you can choose from either the 24- or 96-plex Rapid Barcoding Kits. These PCR-free kits use a transposase to cleave and attach barcodes to your plasmid DNA. Barcoded samples are then pooled and sequencing adapters added. We recommend multiplexing up to 96 plasmid libraries per MinION Flow Cell. Through multiplexing a number of plasmid libraries on a single MinION Flow Cell, the cost per sample can be considerably reduced. Flow cells that are not run at full sample capacity can be washed and reused, facilitating efficient sample batching while maintaining low cost per plasmid. The Flow Cell Wash Kit provides a cost-effective method to wash and re-run a flow cell multiple times. Learn more about Oxford Nanopore library preparation: nanoporetech.com/prepare Barcoded transposome plasmid DNA complex Loading Attachment of sequencing adapters Pooling of barcoded libraries, SPRI clean-up Cleavage of plasmid DNA by transposase and addition of barcodes< 60 min Find more guidance and recommendations for plasmid extraction in our extraction protocols library: nanoporetech.com/extraction-methods We recommend sequencing your plasmid libraries on MinION Flow Cells, which can be run on the portable MinION device for easily accessible, routine sequencing. For consistently running higher sample numbers, the benchtop GridION device enables on-demand sequencing of up to five independent flow cells at one time. For complete, high-quality whole-plasmid sequencing, we recommend basecalling in high accuracy (HAC) mode using the MinKNOW™ software. If you are new to the method, we recommend sequencing for 12 hours, although a shorter run time may be sufficient. De novo assembly and annotation of your plasmids is achieved using the wf-clone-validation workflow. The wf-clonevalidation workflow — an EPI2ME solution — integrates a number of best practice tools for complete plasmid assembly and annotation into an easy-to-use analysis pipeline, including Flye1 for plasmid assembly, Trycycler2 for circularising and refining the assembly, Medaka3 for sequence polishing, and pLannotate4 for annotation. EPI2ME is the user-friendly Oxford Nanopore data analysis platform, suitable for all levels of bioinformatics expertise. The report generated by this EPI2ME workflow presents you with annotated features for each plasmid sequence (e.g. promoters, operators, protein-coding genes), any identified variants in your plasmid sequences compared with a reference, and a multiple sequence alignment between insert sequences from different samples. oxford-nanopore-technologies @nanopore @nanoporetech.com www.nanoporetech.com References: 1. Kolmogorov, M., Yuan, J., Lin, Y., Pevzner, P.A. Nat. Biotechnol. 37(5):540–546 (2019). DOI: https://doi.org/10.1038/s41587-019-0072-8 2. Wick, R.R. et al. Genome Biol. 22(1):266 (2021). DOI: https://doi.org/10.1186/s13059-021-02483-z 3. GitHub. Medaka. Available at: https://github.com/nanoporetech/medaka [Accessed 14 March 2025] 4. McGuffie, M.J. and Barrick, J.E. Nucleic Acids Res. 49(W1):W516–W522 (2021). DOI: https://doi.org/10.1093/nar/gkab374 Sequencing: running until the necessary coverage is achieved Analysis: using the EPI2ME wf-clone-validation workflow Find out more about Oxford Nanopore sequencing devices: nanoporetech.com/sequence View the dedicated EPI2ME workflow: epi2me.nanoporetech.com/workflows/wf-clone-validation View the protocol: nanoporetech.com/plasmid-sequencing-protocol Find out more at: nanoporetech.com/microbiology Information correct at time of publication. May be subject to change. Oxford Nanopore Technologies, the Wheel icon, EPI2ME, GridION, MinION, and MinKNOW are registered trademarks or the subject of trademark applications of Oxford Nanopore Technologies plc in various countries. Information contained herein may be protected by patents or patents pending of Oxford Nanopore Technologies plc. All other brands and names contained are the property of their respective owners. © 2025 Oxford Nanopore Technologies plc. All rights reserved. Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. WF_1146(EN)_V3_28Mar2025
Workflow overview | Whole-plasmid sequencing Rapid and complete plasmid characterisation with Oxford Nanopore sequencing Plasmids are the backbone of molecular biology, playing a pivotal role in applications such as gene therapy and vaccine development, genetic engineering, industrial biotechnology, and basic scientific research. Therefore, routine verification of plasmid constructs is an essential quality control step. Any errors in plasmids, such as mutations, truncations, or rearrangements, can compromise downstream experimental success. Oxford Nanopore sequencing enables the highly accurate, flexible, and secure characterisation of full-length plasmid sequences in-house without the need for primers, with results obtained in hours — negating the need to send constructs to third parties for validation. By obtaining full sequence data in a single experiment, the need for multiple techniques to confirm the identity of your constructs is also no longer required. Here we present a rapid, end-to-end workflow for complete, high-quality whole-plasmid characterisation using a MinION™ or GridION™ sequencing device and the EPI2ME™ analysis platform. Extraction: obtaining high molecular-weight DNA Library preparation: multiplexing samples Selecting an extraction method that will effectively remove contaminants — such as detergents, denaturants, chelating agents, or high salt concentrations — will ensure clean, high-quality DNA samples are taken forward to library preparation. We recommend the use of a plasmid midi prep kit, such as the QIAGEN Plasmid Plus Midi Kit, which enables extraction of reliable quantities of high-purity DNA from overnight cultures. For each sample, 50 ng of plasmid DNA is then taken forward into library preparation. We recommend the Qubit fluorometer for accurate DNA quantification. To prepare your library for sequencing and downstream analysis, you can choose from either the 24- or 96-plex Rapid Barcoding Kits. These PCR-free kits use a transposase to cleave and attach barcodes to your plasmid DNA. Barcoded samples are then pooled and sequencing adapters added. We recommend multiplexing up to 96 plasmid libraries per MinION Flow Cell. Through multiplexing a number of plasmid libraries on a single MinION Flow Cell, the cost per sample can be considerably reduced. Flow cells that are not run at full sample capacity can be washed and reused, facilitating efficient sample batching while maintaining low cost per plasmid. The Flow Cell Wash Kit provides a cost-effective method to wash and re-run a flow cell multiple times. Learn more about Oxford Nanopore library preparation: nanoporetech.com/prepare Barcoded transposome plasmid DNA complex Loading Attachment of sequencing adapters Pooling of barcoded libraries, SPRI clean-up Cleavage of plasmid DNA by transposase and addition of barcodes< 60 min Find more guidance and recommendations for plasmid extraction in our extraction protocols library: nanoporetech.com/extraction-methods We recommend sequencing your plasmid libraries on MinION Flow Cells, which can be run on the portable MinION device for easily accessible, routine sequencing. For consistently running higher sample numbers, the benchtop GridION device enables on-demand sequencing of up to five independent flow cells at one time. For complete, high-quality whole-plasmid sequencing, we recommend basecalling in high accuracy (HAC) mode using the MinKNOW™ software. If you are new to the method, we recommend sequencing for 12 hours, although a shorter run time may be sufficient. De novo assembly and annotation of your plasmids is achieved using the wf-clone-validation workflow. The wf-clonevalidation workflow — an EPI2ME solution — integrates a number of best practice tools for complete plasmid assembly and annotation into an easy-to-use analysis pipeline, including Flye1 for plasmid assembly, Trycycler2 for circularising and refining the assembly, Medaka3 for sequence polishing, and pLannotate4 for annotation. EPI2ME is the user-friendly Oxford Nanopore data analysis platform, suitable for all levels of bioinformatics expertise. The report generated by this EPI2ME workflow presents you with annotated features for each plasmid sequence (e.g. promoters, operators, protein-coding genes), any identified variants in your plasmid sequences compared with a reference, and a multiple sequence alignment between insert sequences from different samples. oxford-nanopore-technologies @nanopore @nanoporetech.com www.nanoporetech.com References: 1. Kolmogorov, M., Yuan, J., Lin, Y., Pevzner, P.A. Nat. Biotechnol. 37(5):540–546 (2019). DOI: https://doi.org/10.1038/s41587-019-0072-8 2. Wick, R.R. et al. Genome Biol. 22(1):266 (2021). DOI: https://doi.org/10.1186/s13059-021-02483-z 3. GitHub. Medaka. Available at: https://github.com/nanoporetech/medaka [Accessed 14 March 2025] 4. McGuffie, M.J. and Barrick, J.E. Nucleic Acids Res. 49(W1):W516–W522 (2021). DOI: https://doi.org/10.1093/nar/gkab374 Sequencing: running until the necessary coverage is achieved Analysis: using the EPI2ME wf-clone-validation workflow Find out more about Oxford Nanopore sequencing devices: nanoporetech.com/sequence View the dedicated EPI2ME workflow: epi2me.nanoporetech.com/workflows/wf-clone-validation View the protocol: nanoporetech.com/plasmid-sequencing-protocol Find out more at: nanoporetech.com/microbiology Information correct at time of publication. May be subject to change. Oxford Nanopore Technologies, the Wheel icon, EPI2ME, GridION, MinION, and MinKNOW are registered trademarks or the subject of trademark applications of Oxford Nanopore Technologies plc in various countries. Information contained herein may be protected by patents or patents pending of Oxford Nanopore Technologies plc. All other brands and names contained are the property of their respective owners. © 2025 Oxford Nanopore Technologies plc. All rights reserved. Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition. WF_1146(EN)_V3_28Mar2025
