Oxford Nanopore Technologies (ONT) offers a method for determining the order of molecules in DNA and RNA. This third-generation sequencing technology directly reads single strands of genetic material, much like a stock ticker tape reveals a message one letter at a time. Unlike previous methods, it analyzes native DNA or RNA without requiring extensive copying or amplification.
The Nanopore Sequencing Process
The core of the technology is a synthetic membrane separating two chambers filled with an ionic solution. Embedded within this membrane are thousands of tiny, barrel-shaped proteins called nanopores. When a voltage is applied across the membrane, it creates a steady electrical current as ions flow through these pores. This stable current serves as the baseline against which all measurements are made.
The sequencing process begins when a motor protein attaches to a DNA strand. This motor protein guides the DNA molecule to a nanopore and begins to unzip the double helix. It then feeds one of the single strands through the protein channel at a controlled speed.
As the single strand of DNA passes through the nanopore, each of the four genetic bases—adenine (A), guanine (G), cytosine (C), and thymine (T)—causes a unique disruption to the ionic current. Each base has a distinct size and shape, obstructing the pore in a characteristic way. This produces a specific electrical signal pattern, sometimes called a “squiggle,” which is measured by an electronic sensor.
These raw electrical signals are streamed to a computer in real time. Sophisticated base-calling algorithms, often powered by machine learning, interpret these characteristic disruptions and translate them back into a sequence of DNA bases.
Unique Capabilities of Long-Read Sequencing
A defining feature is the ability to produce exceptionally long reads of DNA. While older methods break DNA into small fragments, nanopore sequencing can read continuous strands that are tens of thousands of bases long. This is comparable to assembling a puzzle with very large pieces instead of tiny ones, making it easier to see a genome’s complete picture.
The technology also enables real-time analysis of genetic data. As data is streamed directly from the sequencer, scientists can monitor results live and stop the experiment once they have collected enough information. This can shorten the time to an answer from days or weeks to a matter of hours.
The portability of the sequencing devices is another capability. Instruments like the MinION are handheld and can be powered by a laptop, allowing for sequencing outside a traditional laboratory. This has opened up possibilities for on-site genomic analysis in remote field locations, clinics, and even on the International Space Station.
Applications in Science and Medicine
In genomic research, the ability to generate long reads is transforming how scientists assemble genomes. Long reads can span complex and repetitive regions of DNA that are difficult to map with short-read technologies. This allows for more complete and accurate genome assemblies and helps researchers identify large structural variations often missed by other methods.
The speed and portability of nanopore sequencing make it a powerful tool in clinical diagnostics and public health. During infectious disease outbreaks, researchers can rapidly sequence the genomes of pathogens like viruses or bacteria. For example, it was used during the 2015 Ebola outbreak to track the virus’s spread in the field in near real-time. This capability is also applied to monitoring the development of antibiotic resistance.
Environmental science also benefits from on-site sequencing. Researchers can take portable sequencers to remote locations to analyze DNA directly from environmental samples like soil, water, or air. This allows for the rapid identification of microbial communities to assess biodiversity, monitor ecosystem health, or detect invasive species.
Comparison with Short-Read Sequencing
The most apparent difference from short-read sequencing is read length. Nanopore technology generates reads that can be many thousands of bases long, while short-read platforms produce fragments between 50 and 300 bases. This difference dictates the biological questions each technology is best suited to answer.
Historically, a trade-off for long reads was a higher per-base error rate. However, continuous improvements have significantly increased the accuracy of nanopore sequencing. Nanopore sequencing also offers a speed advantage with its real-time data output, providing much faster results than the fixed run times of short-read platforms.
These differences lead to distinct primary uses. Long-read sequencing is ideal for assembling a genome for the first time and for discovering large structural variants. Short-read sequencing remains a strong tool for applications that require counting molecules, like measuring gene expression, or for identifying small genetic variants within an established reference genome.