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Oxford Nanopore Sequencing

Oxford Nanopore Sequencing

Advancements in DNA and RNA sequencing are essential to better our understanding of the fundamental concepts underlying disease, as they provide critical insights into genome biology which can improve our ability to develop novel and effective therapeutics. One of the more recent additions to the sequencing space involves Oxford Nanopore Technologies Ltd., a UK-based biotechnology company, which introduced a unique third-generation, nanopore-based DNA sequencing platform. Rather than using sequencing-by-synthesis chemistry and fluorescent dyes like other competitors, Oxford Nanopore’s technology involves pushing DNA or RNA through a nanopore protein where characteristic disruptions within the electrical current alongside each nucleotide are detected and classified to sequence either DNA or RNA.1

Oxford Nanopore Sequencing Process

The DNA sequencing process occurs when the DNA of interest gets broken up into fragments that range from 500 bp, all the way to the current world record of 2.3M bp long.1,2 Then, adapter sequences are added to the ends of the DNA fragments, in order to serve as a binding site for a motor protein (Figure 1A). The resulting structure is then added to a flow cell that contains an array of tiny nanopores which are embedded into an electro-resistant membrane.3 This membrane is crucial to the entire sequencing platform as it is completely impermeable, thus making the nanopores the only method of transport for ions, as each nucleotide is pushed through.3,4 The motor protein is also crucial for delivering the DNA fragment to the nanopore, as it allows for the unzipping of the double-stranded DNA (dsDNA) fragment, and helps to push it through the nanopore, in order to allow for the sequencing to occur (Figure 1B).4 This is because as each nucleotide is passed through the nanopore, there is a characteristic volume of solution, which contains electrolytes, that passes through the nanopore.3,4 This information is captured due to the fact that each nanopore on the membrane has its own electrode and sensor, allowing the change in electrical data to be collected and analyzed using base-calling algorithms, in order to provide information on the original DNA sequence in real-time (Figure 1C).3

Oxford Nanopore sequencing technology. Mechanism of action

Figure 1: Oxford Nanopore’s DNA library preparation and sequencing process. A. DNA library preparation. Adaptors are added to the target DNA and a motor protein binds and delivers the DNA to a nanopore on the flow cell. B. Motor protein unwinds the dsDNA and pushes it through the nanopore. C. Fluctuations in current as the DNA is pushed through the nanopore are recorded and analyzed.

Third Generation Capabilities and Much More

Oxford Nanopore sequencing falls under the third generation of DNA sequencers, characterized by the ability to directly sequence the target DNA in real-time, in addition to many features of prior technology. One of its most distinctive capabilities pertains to long reads, as Oxford Nanopore’s sequencing technology currently holds the record for the longest read length at 2.3M bp.2,5 Longer read length is ideal for sequencing, as short fragmentation lengths significantly complicate the genome assembly process and are prone to errors when dealing with repetitive and GC-rich regions of DNA.2,5 Given these sources of errors, it is estimated that short-read technology only gives us access to approximately 92-95% of the genome.5 Oxford Nanopore’s sequencing platforms also do not require expensive reagents such as deoxyribonucleotide triphosphates (dNTPs) or DNA polymerases (DNAPs),7 and in some instances, the sample containing the DNA does not require intensive purification prior to sequencing.8 This is an important factor that helps to enable a cost-effective and rapid platform for users to easily and rapidly gain access to desired genetic information. Lastly, pocket-sized devices are also available, bringing DNA sequencing capabilities to the field and into third-world countries that lack access to state-of-the-art laboratories and research facilities.9 

Oxford Nanopore’s Continual Sequencing Improvements 

It is important to note that Oxford Nanopore’s technology was heavily scrutinized for its error rates in the past, which were once as high as 38.2% and many deemed the technology inadequate for use.10 However, the company has since made significant advancements in this aspect, with reports of increasing accuracy in almost every quarter.11 Today, their technology has slightly lower, but comparable accuracy rates to many of the leading sequencing technologies, at 98.3%.11 

Growing Presence on Scientific and Global Communities

For these reasons, Oxford Nanopore’s Sequencing Platform has been utilized for a large number of applications, even though their devices were launched not too long ago, only reaching commercial markets in 2016. Some key areas of study include structural variation, gene expression, identification, splice variation assembly, fusion transcripts and epigenetics. With research areas including microbiology, microbiome, plant and animal biology, human genomics, clinical research, cancer, transcriptomics and infectious disease.12 The technology also continues to play a large role in sequencing and tracking changes in the SARS-CoV-2 viral genome during the COVID-19 pandemic, contributing to approximately 18% of the genomes listed in the global database GISAID.13 In addition, Oxford Nanopore signed its first contract with the UK government, agreeing to provide 450,000 LamPOre tests to National Health Service laboratories for the regular screening of essential front-line healthcare personnel and social workers.13 These recent applications demonstrate Oxford Nanopore’s rapidly growing presence and influence within global scientific communities.

The Future of Nanopore Sequencing Technology

It is important to note that despite possessing 1400+ patents to protect their technology, Oxford Nanopore does not own the exclusive rights to the nanopore technology. As a result, many other companies are currently in the process of developing their own nanopore-based sequencing platforms, including Quantapore and Roche. Regardless, possessing the first-mover advantage in this space serves as a major advantage for the company. In addition, with the ability to offer long read, cost-effective, rapid, and highly accessible sequencing platforms, Oxford Nanopore sets high expectations for future sequencing technologies, whether nanopore-based or not. 


1. Bowden R, Davies R, Heger A, Pagnamenta A, de Cesare M, Oikkonen L, Parkes D, Freeman C, Dhalla F, Patel S et al. Sequencing of human genomes with nanopore technology. Nat. Commun. 2019;10(1).

2. Amarasinghe S, Su S, Dong X, Zappia L, Ritchie M, Gouil Q. Opportunities and challenges in long-read sequencing data analysis. Genome Biol. 2020;21(1).

3. How nanopore sequencing works. Oxford Nanopore Technologies. [accessed 2021 Oct 18].

4. Nanopore Sequencing – How it Works. Oxford Nanopore; 2019.

5. Tørresen O, Star B, Mier P, Andrade-Navarro M, Bateman A, Jarnot P, Gruca A, Grynberg M, Kajava A, Promponas V et al. Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases. Nucleic Acids Res. 2019;47(21):10994-11006.

6. Longer and longer: DNA sequence of more than two million bases now achieved with nanopore sequencing. Oxford Nanopore Technologies. 2018 [accessed 2021 Oct 17].

7. Hofmann N. Nanopore Sequencing Comes to Plant Genomes. Plant Cell. 2017;29(11):2677-2678.

8. Prepare. Oxford Nanopore Technologies. [accessed 2021 Oct 15].

9. MinION. Oxford Nanopore Technologies. [accessed 2021 Oct 17].

10. Laver T, Harrison J, O’Neill P, Moore K, Farbos A, Paszkiewicz K, Studholme D. Assessing the performance of the Oxford Nanopore Technologies MinION. Biomol. Detect. Quantif. 2015 [accessed 2021 Oct 28];3:1-8.

11. Accuracy. Oxford Nanopore Technologies. 2021 [accessed 2021 Oct 16].

12. Applications. Oxford Nanopore Technologies. [accessed 2021 Oct 18].

13. Thomas L. Nanopore technology allows point-of-care SARS-CoV-2 genomic sequencing. News Medical. 2021 [accessed 2021 Oct 18].

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The very first DNA sequencing technology was created by Dr. Frederick Sanger in the 1970s who is often referred to as the ‘father of genomics’ and received a Nobel prize in 1980 for his ground-breaking invention.1 This DNA sequencing technology, called Sanger Sequencing or the Chain Termination Method, works in the following manner. 

Sanger Sequencing & Data Analysis

To prepare the target DNA for sequencing, it is first broken up into short fragments that are denatured into two single-stranded DNA (ssDNA) fragments and a shorter DNA sequence called a primer binds to the ssDNA through complementary base pairing (Figure 1A). Next, DNA polymerase (DNAP) adds deoxynucleotide triphosphates (dNTPs) in solution adjacent to the primer based on complementarity, and this process continues until it reaches the end of the ssDNA. This reaction occurs in four different reaction tubes where each also contains one of the four chain-terminating variations of the dNTPs called dideoxynucleotides (ddNTP) (Figure 1B). Whenever the ddNTPs are incorporated into the elongating strand, DNAP can no longer elongate the DNA strand, terminating the process.2 And since there are many different DNA strands being elongated and terminated in the same test tube, you end up with DNA fragments of varying lengths and molecular weights due to the incorporation of the ddNTP at each nucleotide position on the DNA fragment.2 It is important to note that these ddNTPs are labeled with radioactive molecules which is important for identification as each type of ddNTP has a unique radioactive emission.2

Once the reaction is complete, the DNA fragments are subjected to gel electrophoresis which separates the terminated DNA fragments according to size, where the shortest strands move through the gel the fastest (Figure 1C). The entire gel is then placed under an X-ray or UV light that enables visualization of the radiolabelled ddNTPs.2 Next, by moving up the gel and recording the ddNTP in the sequence, scientists can obtain the sequence of the target DNA molecule (Figure 1C). Given the very limited technology available at the time, the accuracy, robustness, and ease of use made Sanger’s Sequencing one of the most common DNA sequencing technologies used for many years to come.1,2

Figure 1: Sanger Sequencing Technology. A. The target DNA is fragmented, amplified, denatured, and bound to a primer. B. The four reaction mixtures which contain the different radiolabeled ddNTPs where the elongation and termination take place. C. Gel electrophoresis to separate the terminated fragments by size.

The Automation of Sanger Sequencing

Over the following years, Sanger Sequencing experienced several improvements that have allowed for automation of the technology, through the use of fluorescent labels called fluorophores that are bound to the chain-terminating dideoxynucleotides and computers for data acquisition and analysis.3 

Sanger Sequencing & Data Analysis 2.0

Following the same principle and upon completion of the chain-terminating reaction, all the components of this test tube are subjected to gel electrophoresis, which separates the various terminated DNA molecules according to size (Figure 2A). In this improved variation of Sanger’s Sequencing, the gel electrophoresis occurs in a capillary tube, another important aspect of automation (Figure 2B).3 Towards the end of the capillary tube, there is a laser that excites the fluorophore incorporated onto the elongating strand of the DNA molecule to generate a peak that is recorded onto a graph (Figure 2C).3 As a result, this allows scientists to obtain the entire DNA sequence once all the terminated DNA fragments have passed through the laser.

Figure 2: Automated Sanger Sequencing Technology. A. The target DNA is fragmented, amplified, denatured, and bound to a primer. B. The elongation process occurs in a single reaction mixture where the addition of fluorescently labeled ddNTP results in termination. C. Capillary gel electrophoresis to separate the terminated fragments by size and laser excitation for detection.

Sanger Sequencing provided a Launchpad for New and Innovative Technologies

Sanger Sequencing has, for the first time ever, allowed scientists to sequence DNA originating from many organisms which has improved our understanding of pathogens, the origins of disease, and the world around us. Despite what was achieved, its considerable accuracy, and widespread availability have been overshadowed by several outstanding limitations that have bottle-necked its use in modern applications.2,3 This includes how the sequence generated is derived from a single sequencing reaction, as well as the restriction that DNA fragments sequenced must be under 1000 nucleotides long, alongside the fact that the process is highly laborious and time-consuming.2 As a result, Sanger Sequencing has undergone numerous upgrades to improve and keep up to pace with the competition, which is the reason why the automated process is still used today under Thermo Fisher Scientific. However, many of the newer generations of DNA sequencing technologies have, in large part, overtaken Sanger Sequencing. Regardless, this does not discredit the fact that this first-generation DNA sequencing technology paved the way for the subsequent waves of innovative sequencing technologies that have revolutionized medicine.


1. Kchouk M, Gibrat J, Elloumi M. Generations of Sequencing Technologies: From First to Next Generation. Biol. Med. 2017;09(03).

2. Sanger F, Nicklen S, Coulson A. DNA sequencing with chain-terminating inhibitors. PNAS. 1977;74(12):5463-5467.

3. Karger B, Guttman A. DNA sequencing by Capillary Electrophoresis. Electrophoresis.;30(S1):S196-S202.