How Oxford Nanopore sequencing works

Before Oxford Nanopore sequencing technology was available, researchers used traditional sequencing methods to study genomics. First-generation sequencing techniques, such as Sanger sequencing, require amplification of DNA samples and labelling of nucleotides with fluorescent dyes for analysis^1,2. Researchers used this method to sequence the first human genome, but it took many years and resources³.

With the subsequent development of next-generation sequencing (NGS), the human genome could be processed in a few days for less than $1,000³. NGS uses short-read sequencing methods that typically require DNA samples to be fragmented and amplified in fluorescently labelled clusters before the short fragments are computationally reassembled for analysis^1,3.

Oxford Nanopore sequencing is unique in its ability to directly analyse any length of native DNA or RNA — without fragmentation, amplification, or dyes. Oxford Nanopore Technologies was founded in 2005 and released its first nanopore sequencing device in 2014, making real-time analysis of native DNA and RNA accessible. A suite of sequencing devices is now available to suit all experiments — from small portable devices to high-throughput benchtop sequencers.

So, how does Oxford Nanopore sequencing work and how can it benefit your research?

How does nanopore sequencing work?

Nanopore sequencing occurs at the flow cell where nanopores, a tiny pore that allows the movement of molecules through the flow cell membrane, are located. The nanopores pass DNA or RNA through the membrane, which has an electrical current running across it. This means that as a strand of DNA or RNA moves through a nanopore, this electrical current is disrupted. The sequencing software MinKNOW™ then analyses the electrical signal to identify the exact order of bases (including modified bases) that have passed through the nanopore.

What is a nanopore?

Nanopores are small protein channels that naturally exist with various structures and properties. For example, Escherichia coli use nanopores to transport proteins across their membrane. Oxford Nanopore Technologies has engineered nanopore proteins to move DNA and RNA molecules across the membrane in flow cells. Nanopores are located on flow cells, a consumable that you insert into a sequencing device. Once inserted, you can add the DNA or RNA library to the flow cell for sequencing.

Oxford Nanopore Technologies is always researching new nanopore proteins to identify the next best pore for sequencing, ensuring both speed and accuracy are maintained to generate high-quality sequencing data.

How does DNA move through a nanopore?

It takes more than just a nanopore for DNA to be sequenced — an enzyme known as the motor protein is needed to feed a strand through the nanopore. Sequencing adapters — pre-loaded with the motor protein — are attached to both ends of the DNA molecules during library preparation. Once you have added the DNA library to a flow cell, the motor protein associates with a nanopore, then ‘unzips’ the double-stranded DNA and feeds a single strand through the nanopore at a controlled speed for efficient sequencing.

However, sequencing adapters and motor proteins require a little help from tethers to find a nanopore. To prepare the flow cell, you add hydrophobic tethers before the DNA library is loaded, which helps to localise adapted DNA molecules to the membrane, bringing them closer to the nanopores to improve sequencing.

Why is an electrical current used?

As mentioned earlier, the flow cell membrane and nanopores have an electrical current passing through them. When DNA passes through a nanopore, each nucleic acid causes a characteristic change in the current. This electrical signal is known as a ‘squiggle’. MinKNOW records these squiggles, which an algorithm then decodes into nucleotide sequences (a process known as basecalling).

In the video below, watch how nanopore sequencing works at the molecular level.

How are nanopore reads basecalled?

To basecall your squiggle, MinKNOW uses the integrated basecalling software Dorado to decode the squiggle into nucleotide sequences, either during sequencing (known as live or real-time basecalling) or after sequencing (known as post-run basecalling). Once the squiggle is decoded, the reads are stored as BAM or FASTQ files for downstream analysis.

Oxford Nanopore Technologies develops basecallers based on neural networks, a subset of machine learning, to predict nucleotide sequences from the electrical signal. The basecalling algorithms are trained on known datasets to help the model correctly predict bases and minimise errors during basecalling to increase accuracy. Training datasets include a range of native and amplified DNA and RNA from different organisms, as well as native nucleic acid sequences with specific base modifications to finetune modification-aware models.

Oxford Nanopore Technologies is always developing new and improved basecalling models and new releases can be found on the Oxford Nanopore Github as open-source tools.

How long is an Oxford Nanopore sequencing run?

Oxford Nanopore sequencing runs can be as long or as short as you want because data is streamed in real time, allowing you to monitor a sequencing run, and giving you the control to stop sequencing as soon as enough data is generated for your analysis. This can be anywhere from under an hour, all the way up to 72 hours or when the nanopores on a flow cell are exhausted.

Real-time data streaming is possible because as soon as a strand of DNA or RNA enters a nanopore, MinKNOW can begin basecalling the electrical signal before the strand has even finished passing through a nanopore — giving you immediate access to data.

For sequencing devices with onboard compute (GridION, PromethION 2 Integrated, and PromethION 24), live basecalling is compatible with modification calling, barcode demultiplexing, and alignment to a reference genome. With the availability of these real-time data analysis tools, specifically live alignment, Oxford Nanopore Technologies (with innovation from the Nanopore Community^4–7) has developed a unique targeted sequencing method: adaptive sampling. This method provides software-based enrichment and depletion of regions of interest during sequencing, with no additional library preparation needed. For more information, keep an eye out for an adaptive sampling blog coming soon!

How can Oxford Nanopore sequencing benefit my research?

In summary, through the combination of nanopores, motor proteins, an electrical current, and basecallers, Oxford Nanopore Technologies gives you access to analyse the DNA and RNA of anything, anywhere through direct sequencing of nucleic and ribonucleic acids. Furthermore, the complete process of nanopore sequencing is fast and simple because fragmentation, amplification, and dyes are not required.

There are also many other benefits of Oxford Nanopore sequencing, including the analysis of any fragment length, real-time data streaming, and base modification analysis. This means that nanopore technology can be — and has been — used across a wide range of applications, from microbiology and population genomics to human translational and cancer research.

There are many more features available from Oxford Nanopore Technologies that will be covered in future posts and will be available soon on the blog page. In the meantime, browse the Resource Centre to discover how your fellow scientists have used Oxford Nanopore sequencing to answer their research questions.

Find out what you can discover with the Oxford Nanopore all-in-one platform solution.

Stephanie Chrysanthou presenting at the 2024 Nanopore Community Meeting in Boston, demonstrating how Oxford Nanopore sequencing and adaptive sampling transformed her targeted sequencing workflow to rapidly call cancer-associated pathogenic variants.