Oxford Nanopore Technologies (ONT) sequencing is a third-generation genomic technology based on electronic detection. It transforms the process of reading genetic material into a direct, real-time electrical measurement. By utilizing microscopic protein channels, ONT makes sequencing DNA and RNA quickly and affordably. This technology has fundamentally changed where sequencing can be performed, making it accessible outside of large, centralized laboratories.
The Core Mechanism of Nanopore Technology
The technology is founded on the use of a nanopore, a minuscule channel often made of a modified protein, embedded within an electrically resistant synthetic membrane. This membrane separates two fluid-filled chambers containing an electrolyte solution. An electrical voltage is applied across the membrane, which drives a steady stream of ions through the nanopore, creating a measurable ionic current.
A DNA or RNA strand is prepared with a specialized adapter that includes an attached motor protein. This motor protein binds to the nucleic acid molecule and acts like a molecular brake, controlling the rate at which the strand passes through the nanopore. As the nucleic acid molecule translocates through the pore, the chain of nucleotides physically obstructs the channel, disrupting the flow of ions.
Each combination of bases (a group of about five nucleotides, known as a k-mer) that sits within the nanopore causes a unique change in the ionic current. An application-specific integrated circuit (ASIC) monitors these momentary disruptions in the electrical signal. Algorithms then translate the sequence of electrical changes into the corresponding sequence of A’s, T’s, C’s, and G’s, effectively reading the genetic code. This direct electrical reading of the native molecule differentiates nanopore technology from older sequencing methods that require chemical termination or light-based detection.
Key Advantages of ONT Sequencing
The direct electrical reading mechanism provides several benefits that expand the potential applications of sequencing. Since the nucleic acid molecule is threaded continuously through the pore, the technology generates exceptionally long contiguous sequences, often reaching tens to hundreds of kilobases and into the megabase range. These ultra-long reads are useful for assembling complex genomes, resolving highly repetitive regions, and identifying large-scale structural variations that short-read technologies cannot span.
The ability to analyze data in real time is another key feature, often called “sequencing on demand.” Electrical signals are processed and base-called as they are generated, eliminating the need to wait for an entire batch run to finish. This allows researchers to monitor data quality mid-run and make immediate decisions, such as stopping a run early or using adaptive sampling to focus sequencing on molecules of interest.
The physical design of the MinION device, which is pocket-sized and USB-powered, provides unparalleled portability. This small form factor allows high-quality sequencing to be performed outside of traditional laboratories, such as in remote field locations or during infectious disease outbreaks. Sequencing native DNA or RNA molecules directly also allows for the simultaneous detection of epigenetic markers, like DNA methylation, without additional chemical preparation steps.
Scaling Up: The Range of ONT Devices
The core nanopore technology is deployed across a range of hardware platforms, offering scalability from single-experiment use to massive population-level studies.
Portable and Low-Throughput Devices
The Flongle and MinION devices represent the portable, low-throughput end of the spectrum, used for rapid, on-site sequencing. The Flongle uses a small, single-use flow cell capable of generating up to 2.8 gigabases (Gb) of data, ideal for rapid quality checks or small, targeted experiments. The MinION is a highly portable device that yields up to 50 Gb per flow cell, making it a standard tool for field research and smaller projects.
Mid-Sized Benchtop Systems
Scaling up from the portable devices is the GridION, a compact benchtop instrument. It can house up to five MinION-compatible flow cells simultaneously, acting as a modular system. This allows for multiple independent experiments to run concurrently, generating a throughput of up to 250 Gb in a single run. The GridION is well-suited for mid-sized laboratories requiring flexibility and increased data output.
High-Throughput Institutional Systems
For projects demanding the highest throughput, the PromethION system is designed for large-scale institutional sequencing. The PromethION 24 and 48 models run up to 24 or 48 high-capacity PromethION flow cells, respectively. Each PromethION flow cell produces up to 290 Gb of data. The largest system can generate terabases of data for projects like whole-human-genome sequencing or large population genomics studies.
Real-World Scientific Impact
The portability and speed of nanopore sequencing have led to its adoption where rapid turnaround time is beneficial, affecting global health and environmental monitoring. In infectious disease, the technology enables real-time genomic tracking of outbreaks. For instance, the MinION was deployed during the 2015 Ebola outbreak in West Africa, allowing researchers to sequence the virus genome and track its evolution and transmission in the field within hours, rather than weeks.
This capability was also used during the SARS-CoV-2 pandemic, where sequencing in local labs globally helped identify new variants and monitor their spread quickly. The ability to rapidly identify pathogens from complex samples, such as those from patients with suspected pneumonia, has also proven valuable in clinical settings. Metagenomic sequencing using ONT shows pathogen detection rates comparable to conventional culture methods, but with a much faster turnaround time, often identifying a causative agent within hours.
Beyond human health, the technology is applied in environmental and ecological research. Its portability allows scientists to sequence DNA directly at remote sample sites, which is beneficial for monitoring biodiversity in challenging locations. This field-based sequencing facilitates rapid species identification and ecological surveys, providing immediate data that can inform conservation efforts or detect invasive species. The technology’s flexibility and ease of deployment have pushed the boundaries of genomic science out of the specialized core facility and into the real world.