In the current sequencing landscape, two major technologies are often used to read the nucleotide bases in plasmid DNA: the traditional Sanger method and long-read, 3rd generation sequencing using either Pacific Biosciences (PacBio) or Oxford Nanopore Technology (ONT) nanopore sequencers.
These two methods have a few fundamental differences and work best when used synergistically for an accurate and comprehensive view into the complete plasmid sequence. In this article, we will explore the cutting-edge realm of genomic research, and how this innovative NGS technology offers real-time, long-read capabilities.
What Is 3rd Generation Sequencing?
Third-generation sequencing generates high-quality long reads with amplification-free library prep. This method improves on PCR-based amplification, which can introduce unwanted artifacts. The DNA strand is separated, enters the nanopore protein, and an electric current runs through the single-stranded (long) DNA, measuring the nucleotides in real time.
Once you are satisfied with the amount of data that is generated to produce the accuracy you desire, you can stop the nanopore run. There is no set limit on cycles. Results are generated and uploaded rapidly to your local server or cloud computing to complete your bioinformatic analysis.
How Does 3rd Generation Sequencing Differ from Sanger Sequencing?
Sanger sequencing is well-suited for plasmid sequence validation due to its accuracy in reading short DNA fragments. It is a reliable method to focus on specific regions of the plasmids, such as gene insert sequences that often need to be validated.
Unlike Sanger sequencing, 3rd generation sequencing platforms excel in generating exceptionally long reads (e.g. 30 kb to 1 Mb), making it easier to capture complete plasmid sequences with just a few cycles of sequencing. This capability is invaluable when dealing with complex plasmid structures, allowing for a comprehensive understanding of gene arrangements, regulatory elements, and other critical features.
Additionally, 3rd generation sequencing offers the ability to multiplex samples according to the flow cell size/capacity and the expected output for the analysis. By contrast, Sanger is a single sample per well sequencing assay. Total read length is limited to 800 bp per sample target.
Sample preparation for plasmid sequencing with 3rd generation sequencing is streamlined without the use of primers, reducing labor-intensive steps associated with traditional Sanger methods.
While 3rd generation sequencing offers strong advantages in read length and real-time monitoring, there are a few potential technical challenges. Higher error rates, particularly in homopolymeric regions, make it less than ideal for sequence confirmation with complete accuracy.
Therefore, proper sample coverage is required to increase the consensus calling of the DNA molecule. Otherwise, it's recommended to leverage both 3rd generation sequencing and Sanger together for the best results, while minding budgetary considerations.
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Sanger Sequencing |
3rd Generation Sequencing/ONT |
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Accuracy |
Low error rate. High accuracy method.
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Higher error rates, particularly in homopolymeric regions. O |
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Read Length |
Shorter reads (typically ~800-1000 bp reads). |
Longer reads (typically 10kb - 1Mb or higher, however smaller reads down to 500 bp are possible). |
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Sample Preparation |
Often requires multiple reactions, involving tedious cloning and DNA amplification, designing new primers for each reaction. |
Streamlined sample preparation, reducing time and complexity of workflows. |
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Throughput and Cost |
Lower throughput, single sample per well. Higher cost per base. |
Higher throughput – multiplex samples per well (typically >96 samples per chip). Lower cost per base.
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Primers |
Requires primer design to amplify regions of interest. |
No primer design necessary, entire plasmid can be sequenced at once. |
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Applicability for Whole Plasmid Sequencing |
Precise and reliable for short sequences; well-suited for validation of specific regions within the plasmids. |
Suitable for generating full plasmid sequence and annotation maps. |
Why Psomagen Uses Oxford Nanopore for Whole Plasmid Sequencing (WPS)
Psomagen uses Oxford Nanopore (ONT) sequencers to perform whole genome sequencing on plasmids. ONT’s nanopore workflow for WPS offers the best value to our customers for the dataset that’s produced for the application.
Compared to Sanger sequencing which offers shorter reads (typically less than 1000 bp), ONT provides a far more efficient way to sequence longer DNA strands (with reads typically 10kb or higher), higher throughput through multiplexing, and low cost per base read. Our WPS service offers a rapid turnaround time due to Psomagen’s streamlined sample preparation, rapid sequencing, and integrated bioinformatics which contribute to a complete solution for our customers. While Sanger sequencing has been a gold standard for validating short sequences of specific regions, 3rd generation sequencing methods generate long-read, full plasmid sequences and annotation maps. However, Psomagen does offer the ability to perform plasmid sequencing by either method – nanopore sequencing is the best scientific and economical solution for our customers.
How should I prepare my sample for submission?
Getting to a purified plasmid takes several steps. Psomagen’s whole plasmid sequencing is carried out by:
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isolating the plasmid DNA (pDNA),
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splicing the pDNA into smaller fragments,
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sequencing those fragments, then
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assembling the plasmid sequence data to reconstruct the full plasmid genome.
Our bioinformatic tools then analyze the resulting sequencing data to provide a comprehensive look at your plasmid. These results include including consensus plasmid assembly and plasmid annotation files. The full workflow is depicted below:
Plasmid construction workflow
Visit our Sample Submission Guidelines for info on the size, minimum amount, concentration, purity, and buffer for best sequencing results. Send us your sample for high-quality results in approximately 14 hours. WPS results include FASTQ files (raw data of 3rd Gen. NGS), read-length and quality scoring, consensus plasmid assembly, and annotation.
Limitations of 3rd Generation Sequencing / ONT for Whole Plasmid Sequencing
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The sample must meet a 50 ng/μL concentration. Low concentration causes an increase in fragmentation and a low number of reads.
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Poly A and homopolymer regions may be truncated. In this case, users would benefit from follow-up Sanger sequencing for that specific region. You can discuss these specific needs with your Psomagen specialist.
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ONT is set up for clonal populations. It is not suitable for sequencing multiple plasmids in a mixed sample.
What Are Key Factors in Choosing a Plasmid Sequencing Technology?
These two questions can help you make an educated decision in choosing a plasmid sequencing technology:
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Where do you expect to find relevant genetic information? If you’re growing and isolating individual colonies and looking at several mutations throughout the entire plasmid, Psomagen’s WPS using Oxford Nanopore will be the best solution. If you are creating and analyzing a small mutation, such as 500 base pairs, our Sanger services are recommended.
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Does your experiment include random mutants? If your sample contains a mixture of different mutations (not growing out colonies), a variant analysis with NGS would be advised.
When and How Do Sanger and 3rd Gen Sequencing Complement Each Other?
While ONT offers strong advantages in read length and real-time monitoring, we’ve mentioned a few potential challenges including higher error rates, particularly in homopolymeric regions, which make 3rd Gen less than ideal for sequence confirmation with complete accuracy. That’s where Sanger sequencing can complement ONT — since Sanger has higher read accuracy for short DNA fragments, Sanger is particularly well-suited for validating challenging regions, such as gene insert sequences, of a plasmid sequence.
By knowing some of the limitations of your plasmid sample and the capabilities of ONT, you can attain the best turnaround time and lowest cost by using a combination of the two services. Your Psomagen project management team can provide recommendations to address your specific research needs for your construct, including aligning both whole plasmid sequencing data and Sanger data.
Why Customers Choose Psomagen for Whole Plasmid Sequencing
Psomagen deploys 3rd generation sequencing for our WGS service. This technology delivers cost-effective, efficient, and streamlining genomic interpretation with rapid turnaround time, seamlessly integrating into your experiments.
Psomagen is a US-based contract service lab, meaning your samples stay in the U.S. We have easily accessible sites in Maryland, New York, and Boston. Not located in one of those geographies? Place your order, and we’ll send you a shipping label to ship your samples overnight.
Finally, customers experience market-leading pricing from Psomagen, meaning great value for your results.
About the Author
Paul Kim has been a key member of the Psomagen team for 9 years,
bringing his extensive experiences in Sanger and Nanopore sequencing to our facilities. Inside the lab and out, he provides advice and
support to various projects and clients. Paul's proactive approach to sharing his experiences and his commitment to ongoing learning has made him a highly valued part
of the Psomagen family.
