Nanopore sequencing is a method for decoding the genetic material, including DNA and RNA, of living organisms. This third-generation technology identifies the sequence of this material by passing it through a microscopic pore, offering a new way to access biological information.
Imagine identifying the individual beads on a long string by pulling it through a tiny hole that senses each bead as it passes. Nanopore sequencing operates on a similar principle, decoding the fundamental components of life one by one. This technology has transformed how scientists investigate everything from viruses to complex human genomes.
How Nanopore Sequencing Works
The core of nanopore sequencing combines biology and electronics, centered around a nanoscale pore. These pores are proteins that form a channel, embedded within an electrically resistant synthetic polymer membrane. This assembly is submerged in an ionic solution, and when a voltage is applied across the membrane, it generates a steady flow of ions through the nanopore, creating a measurable electrical current.
To read a DNA or RNA molecule, it is guided to and threaded through a nanopore. A motor protein, attached to the nucleic acid strand during sample preparation, latches onto the nanopore. The motor protein unwinds double-stranded DNA into a single strand and ratchets it through the pore at a controlled speed for accurate measurement.
As the single strand of DNA or RNA moves through the nanopore, each nucleotide base—adenine (A), guanine (G), cytosine (C), and thymine (T) in DNA, or uracil (U) in RNA—obstructs the pore. Because each base has a distinct size and chemical structure, it causes a characteristic disruption in the ionic current. The device’s sensors detect these changes in the electrical signal in real time.
Unique Capabilities of Nanopore Technology
Nanopore sequencing has several distinct capabilities that set it apart from other methods.
- Unlike previous technologies that generate short DNA fragments of a few hundred bases, nanopore sequencers can read continuous strands that are tens or even hundreds of thousands of bases long. Assembling a genome from short reads is like trying to solve a massive puzzle with tiny, almost identical pieces, especially in repetitive regions. Ultra-long reads are like having very large puzzle pieces, making it much easier to see the big picture and correctly assemble complex parts of a genome.
- Data becomes available for analysis the moment the sequencing run begins, streamed directly from the device. This allows researchers to monitor the results as they are generated and even stop the experiment once a particular finding has been made. This “Read Until” function allows the device to be programmed to eject molecules that are not of interest and select others, optimizing the efficiency of the sequencing run for targeted applications.
- The technology’s portability has broadened the horizons of genomic research. Devices like the MinION are palm-sized, weigh under 100 grams, and can be powered by a laptop’s USB port. This portability allows for in-field sequencing, taking the laboratory directly to the sample source, whether that’s a remote clinic during an outbreak or an environmental site in the Amazon rainforest.
- Nanopore sequencing can directly analyze native DNA and RNA molecules without the need for amplification, a step required by many other methods. This avoids biases that can be introduced by PCR amplification and preserves natural modifications on the bases, such as methylation. These epigenetic markers, which can be detected simultaneously with the nucleotide sequence, provide an additional layer of biological information that is often lost with other technologies.
Applications Across Science and Medicine
In public health, the portability and real-time analysis of nanopore sequencing support tracking infectious disease outbreaks. During the Ebola, Zika, and COVID-19 epidemics, portable sequencers were deployed to remote locations to rapidly identify viral strains, monitor their evolution, and understand transmission patterns directly at the source.
In clinical and cancer research, the ability to generate long reads is transforming our understanding of complex genetic diseases. Many cancers are characterized by large-scale structural rearrangements in the genome, such as translocations or inversions, which are difficult to detect with short-read sequencing. Nanopore technology can span these complex regions, providing a clearer picture of the genetic changes that drive cancer. It also aids in the diagnosis of rare genetic disorders caused by large structural variants that were previously challenging to identify.
Environmental and ecological studies have also benefited from portable nanopore sequencing. Scientists can now perform genomic analysis in the field, far from a traditional laboratory setting. For instance, researchers use nanopore sequencers to identify species by analyzing environmental DNA (eDNA) from soil or water samples. This technique has been used to monitor biodiversity in ecosystems ranging from the North Sea to remote rivers, detecting everything from fish species to invasive parasites and pathogens.
The technology is also finding use in agriculture and food safety. It can be used to sequence plant genomes, which are often large and complex, helping to improve crop breeding. In food safety, the rapid and portable nature of nanopore sequencing allows for the quick identification of pathogens contaminating the food supply chain or for verifying the origin of food products.
From Electrical Signal to Genetic Code
The raw data from a nanopore sequencer is not a sequence of letters but a continuous, fluctuating electrical signal called a “squiggle.” This signal represents the changes in ionic current as a DNA or RNA strand passes through the pore. The process of converting this analog signal into a digital sequence of nucleotides is a computationally intensive step known as “basecalling.”
Basecalling relies on sophisticated algorithms, which have increasingly incorporated machine learning and neural networks. These algorithms are trained on vast datasets of known DNA sequences to learn the distinct electrical signature produced by each nucleotide, or more accurately, short sequences of nucleotides (k-mers). The software analyzes the raw squiggle data in real-time, identifies the characteristic disruptions, and translates them into a base sequence stored in a standard format like a FASTQ file.
A historical challenge for nanopore sequencing was its higher per-base error rate compared to short-read technologies. Accuracy has improved dramatically over the years due to advancements in the nanopore protein itself, improved chemistry, and more refined basecalling algorithms. Newer pores, for instance, may have two “reader” sections instead of one to improve accuracy.
Current raw single-read accuracy is now greater than 99%. While individual read accuracy may sometimes be lower than that of short-read methods, the ability to sequence very long strands provides a major advantage. By sequencing the same DNA molecule multiple times or by generating a consensus from many different long reads covering the same region, a highly accurate final sequence can be achieved, with accuracy that can exceed 99.9%.