How Oxford Nanopore sequencing works

What is a nanopore?

Nanopores are tiny protein channels that occur naturally in biological membranes, where they allow molecules to pass in and out of cells. Oxford Nanopore has engineered nanopore proteins to analyse DNA and RNA molecules as they pass through the pore. Thousands of nanopores are embedded in an electrically resistant membrane within a flow cell — the consumable inserted into an Oxford Nanopore sequencing device.

How does DNA or RNA pass through a nanopore?

During library preparation, sequencing adapters pre-loaded with a motor protein are attached to the DNA or RNA molecules to be sequenced. When the prepared library is loaded onto a flow cell, the motor protein associates with a nanopore and controls the speed at which the molecule passes through the pore. A voltage applied across the membrane creates an electric field that draws DNA or RNA through the nanopore, while also generating an ionic current through the pore.

For double-stranded DNA, the motor protein separates the two strands before feeding a single strand through the nanopore. RNA can be sequenced directly as a single strand.

How are the bases identified?

As a DNA or RNA molecule passes through the nanopore, different combinations of bases cause characteristic changes in the ionic current. These electrical signals are measured and interpreted by basecalling algorithms to determine the nucleotide sequence.

Because each molecule is read continuously as it passes through the nanopore, read length is only limited by the length of the DNA or RNA presented for sequencing, rather than by a fixed platform limit. And because Oxford Nanopore sequencing does not require amplification, native DNA and RNA can be analysed directly, retaining base modifications (e.g. methylation) that can be individually identified through changes in the signal.

Together, these capabilities enable you to capture richer biological information using a single sequencing technology.

Basecalling and modification detection can be performed in real time via MinKNOW, the software that controls all Oxford Nanopore sequencing devices.

Real-time basecalling also powers Adaptive Sampling, our unique on-device targeted sequencing method, enriching regions of interest as sequencing happens — with no prior wet-lab enrichment required.