Plasmid Sequencing in 2025

Why Plasmid Sequencing

Plasmid sequencing is a critical step in confirming successful cloning and plasmid construction. Verifying plasmids before use is essential, as they can acquire mutations, be edited, or get mislabeled over time. If you’re getting plasmids from third parties, sequencing ensures the genetic content matches its published specifications. For minimal cost, plasmid sequencing provides minimizes the risk of costly and time-consuming experimental failures. This is especially important when plasmids are starting materials in biomanufacturing processes, where even minor errors can lead to liability exposures and product recalls.

Whole Plasmid Sequencing or Insert Sequencing

Traditionally, researchers only sequenced the gene inserted in a plasmid, assuming the backbone was unchanged and unimportant. However, next-generation sequencing (NGS) advances have made it practical and cost-effective to sequence entire plasmids. This comprehensive approach validates all components, including the origin of replication, selectable markers, and regulatory elements, ensuring they are intact and free of unintended mutations.

Whole-plasmid sequencing is particularly crucial in fields like gene therapy and synthetic biology, where even a tiny error in the plasmid can have significant implications for functionality or safety. It also supports a more holistic approach to genetic design, including genes of interest and the genetic elements controlling their expression.

Sequencing Technologies

Today, scientists have access to three different sequencing technologies that complement each other.

Sanger sequencing is ideal for confirming the sequence of genes inserted at the plasmid cloning site. Many plasmids include primer binding sites on both sides of the cloning site, which makes it possible to use standard sequencing primers. Sanger sequencing has been the gold standard of plasmid sequencing for many years. This technology has two main advantages. It provides very good quality data that can be analyzed without bioinformatics skills. It is also possible to perform individual sequencing reactions at a very affordable price. However, the technology also has significant limitations. First, the design of primers requires some knowledge of the plasmid sequence. Sequencing fragments longer than 1,500 bp requires the design of custom primers that need to be ordered from an oligo synthesis company, complicating the sequencing process and increasing its cost. Finally, the small amount of data produced by individual sequencing reactions quickly adds up, making whole plasmid sequencing expensive and impractical. Despite these limitations, Sanger is still very popular. Applied Biosystem’s SeqStudio is a capillary electrophoresis system that makes it easier than ever before to perform Sanger sequencing internally.

Nanopore sequencing is a long-read sequencing technology developed by Oxford Nanopore.  It excels in mapping entire plasmids, detecting structural variations, and resolving repetitive regions. Its portability and scalability make it versatile for both research and industrial use. This technology is attractive because it does not require expensive instruments. The MinION is a small, inexpensive USB device that allows individual labs to perform nanopore sequencing. Another advantage of this technology is that the library preparation process is simpler and cheaper than other NGS technologies. However, nanopore sequencing has higher error rates than short-read technologies, particularly in homopolymeric regions or areas with complex secondary structures. Careful analysis and error correction are often necessary to ensure accuracy in critical applications.

Short-read sequencing is a high-throughput sequencing technology that can sequence plasmids, even though it is mainly geared toward genomic applications. The Illumina MiSeq is an entry-level sequencer well-suited to sequence plasmid libraries. It delivers large volumes of high-quality data that can achieve base-level accuracy with most plasmids. Operating an Illumina sequencer can be costly. The cost structure is complicated to decipher. The technology is best suited for high-throughput facilities that can include many plasmids in a single run. In addition, many vendors like SeqWell and QuantaBio provide library preparation kits compatible with Illumina technology. These kits compete with the chemistries sold by Illumina, but they require some investment in process development. 

Combining sequencing technologies

GenoFAB has pioneered hybrid plasmid sequencing that combines data from MinION and Illumina technologies. This integrated approach ensures complete and accurate plasmid validation by leveraging the structural insights of long reads with the base-level precision of short reads. The hybrid method minimizes the risk of undetected errors and is particularly advantageous for plasmids used in high-stakes applications like biomanufacturing, where absolute accuracy is critical.

While one might assume that precisely controlling the sequencing process would result in consistent and reproducible outcomes, whole plasmid sequencing with NGS involves several steps that can introduce variability. Surprisingly, some plasmids are inherently challenging to sequence due to factors like their structure or sequence composition. These challenges highlight the importance of careful design considerations for genetic engineers when constructing plasmids. For more details on sequencing variability, see this study.

Bioinformatics Analysis of Sequencing Data

Generating high-quality sequencing data is only part of the process—interpreting it accurately is equally essential. GenoFAB has collaborated with the Colorado State University Biofoundry to develop an open-source bioinformatics pipeline to efficiently assemble plasmid sequencing data. They also developed a web interface allowing labs with limited bioinformatics support to analyze their sequencing data. By converting raw data into actionable insights, the pipeline streamlines decision-making for both research and production settings.

Plasmid Sequencing Service

GenoFAB offers comprehensive plasmid sequencing services tailored to meet a variety of research and industrial needs. Whether you require structural verification, base-level precision, or advanced bioinformatics analysis, GenoFAB delivers high-quality results that you can trust. Our services are designed to support a wide range of applications, from academic research to commercial biomanufacturing. With flexible options and volume discounts, we accommodate projects of all sizes while maintaining the highest standards of quality.

Conclusion

Plasmid sequencing is an often-overlooked cornerstone of modern molecular biology and biotechnology. By leveraging advanced sequencing technologies and robust bioinformatics pipelines, GenoFAB ensures that your plasmids meet the highest quality standards. This enhances the reproducibility of downstream experiments and accelerates R&D projects by reducing the risks of errors that could derail critical experiments or processes.

Integrating sequencing in plasmid development workflows proves challenging for many organizations. GenoFAB end-to-end plasmid management services relieve scientists who need these plasmids from the burden of preparing plasmid solutions, ordering sequencing, and performing their bioinformatic analysis.  

Replies to comments

• @John: The two methods have different error types. Illumina is more accurate than Nanopore on most sequences. Confirming the sequence of specific regions with Sanger is another option if the sequence requiring confirmation is small enough. It quickly becomes expensive when multiple reads are necessary. The best combination depends on the number of plasmids that need to be sequenced and the goal of sequencing. Someone using plasmids as starting material for a cGMP manufacturing process has a different standard than someone seeking to identify a plasmid or verify its overall structure. That is why we are offering different tiers of sequencing services.