What is Oxford Nanopore Technologies (ONT) sequencing?

• In 2015, the first nanopore sequencing device was released by Oxford Nanopore Technologies – an entirely new way to sequence DNA.
• Unlike previous techniques, which were based on DNA replication, ONT doesn’t use any DNA polymerases. Instead, it’s based on a small barrel-shaped protein in a membrane, called a nanopore, and measures changes to electrical current.
• It was used in 2015 during an Ebola outbreak to help with genomic surveillance.

What is nanopore sequencing?

• Nanopore sequencing is a type of third generation sequencing technology from ONT.
• Unlike all previous sequencing technologies, ONT doesn’t use any DNA polymerases.
• Instead, it uses a barrel-shaped protein called CsgG – naturally found as a ‘pore’ in a cell membrane, regulating which molecules can enter or leave a cell. This is called a nanopore.
• CsgG has a diameter of 1 nanometre at its smallest point – just big enough to allow a single strand of DNA through.

How does nanopore sequencing work?

• In nanopore sequencing, the CsgG nanopore is embedded into an artificial membrane inside a sequencing chamber. When an electrical current is applied across the membrane, the current has to flow through the nanopore.
• DNA is threaded through the nanopore by an enzyme - a molecular motor - and it obstructs the current flowing through the nanopore.
• The four bases of the DNA (A, T, C and G) are of different shape and size, so cause characteristic variations in the current.
• These variations are measured by an electronic chip. An algorithm converts the data into a sequence which can then be read.

What are the benefits of nanopore sequencing?

• It can generate both very short (20 bases) and ultra-long reads (up to several millions of base pairs) making it much easier to sequence an entire genome.
• The instrument is very small – depending on the model, between the size of a phone and a microwave. Comparatively, Illumina and PacBio sequencers are closer in size to fridge-freezers. ONT’s small size makes the technology more accessible outside of the traditional laboratory setting, requiring only a laptop to run it.
• For example, the smallest ONT sequencer - the MinION - has been used onboard the International Space Station. It has also been used in low-income countries, including during the Ebola pandemic in 2015 – when it read the genomes of Ebola viruses from 14 patients in just 48 hours.
• The biggest ONT sequencer - the PromethION - can be used for human genome sequencing and was vital for filling in the gaps when the human genome was finally complete in 2021.