What is Oxford Nanopore Technology (ONT) sequencing?

How does ONT work?

• ONT is based on a completely different principle than PacBio and Illumina technologies as it doesn’t use any DNA polymerases in its instruments.
• ONT uses another type of naturally occurring protein, the pore-forming protein α-hemolysin. These barrel shape proteins are typically found embedded within cell membranes and regulate which molecules can enter or leave the cell.
• α-hemolysin happens to have an inner diameter of 1 nm, just large enough to allow a single strand of DNA through.

Nanopore proteins are embedded into an artificial membrane inside the sequencing flow cell. A current is applied across the membrane forcing the negatively charged DNA strands to move through the nanopores. The obstruction of a nanopore by a DNA fragment leads to a change in the current that is measured continuously by an electronics chip integrated within the flow cell. A base-calling algorithm converts the current variation back into the original DNA sequence.
Image credit: Genome Research Limited.

How is ONT sequencing controlled?

• ONT flow cells are controlled by an electronic chip that sits just under an artificial membrane containing hundreds or thousands of these protein nanopores.
• A voltage is set across the membrane attracting negatively charged DNA molecules through the nanopores, obstructing the current across the membrane.
• Because the 4 bases of DNA have different shapes and sizes, they lead to different current variations when moving through a nanopore.
• These current variations make up ONT’s raw data.
• A base-calling algorithm then converts the current variation traces into a sequence, each intensity step corresponding to the multiple bases obstructing the pore at a certain time.

Why use ONT?

Despite having a lower accuracy than some of the other technologies, ONT offers several unique advantages.

• First of all, as long as a DNA sample is handled carefully, reads can reach several millions of base pairs. These ultra-long reads significantly decrease the number of jigsaw puzzle pieces when assembling the sequencing data into a genome.
• Depending on the model, the size of an ONT instrument ranges between that of a mobile phone and microwave. For comparison, Illumina and PacBio largest sequencers are similar to large fridge-freezer units. Why is this an advantage?
  • Large sequencers offer greater throughput, but a small portable device requiring nothing more than a powerful laptop can be key to performing experiments outside of the classical academic laboratories.
  • ONT’s smallest sequencer has been used in low-income countries, notably during the Ebola pandemic in 2014, in Antarctica, or even on board the International Space Station!

MinION sequencing device connected to a laptop
Image credit: Oxford Nanopore Technologies

MinION sequencing device being used in Guinea during the 2013-2016 Ebola outbreak.
Image credit: Tommy Trenchard / European Mobile Laboratories

Article written by Louise Aigrain, Senior Staff Scientist in the DNA Pipelines Research and Development team” at the Wellcome Sanger Institute.