What is Oxford Nanopore Technology (ONT) sequencing?

Oxford Nanopore Technology developed third generation sequencers that are portable, able to sequence DNA in remote locations and produce ultra-long reads.

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.

This page was last updated on 2021-12-14

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