New sequencing technologies are largely funded with the hope that the next technology will take sequencers into the diagnostics world. This requires overcoming two present hurdles: cost per base and turnaround time. At the Advances in Genome Biology and Technology conference held at Marco Island, 2012, Oxford Nanopore Technologies introduced its USB size sequencer ‘MinION’, and their bench top sequencer ‘GridION’.
Two sequencers and a technology that is not yet commercially available but potentially very exciting. James Hadfield introduced us to Oxford Nanopore Technology’s ‘MinION’ in his third and final part on the history of sequencing on this Channel.
But in this article we’ll look at the principle of nanopore sequencing, its challenges and decide if the sequencers currently developed at Oxford Nanopore Technology will find a way around it.
Is there a bottleneck in the existing technologies?
All the sequencers available right now require sample amplification leading to increase in prep time and cost. The promise of nanopore sequencing is to eliminate amplification (along with the errors that it brings) and instead use what is called as ‘Single molecule sequencing’. The concept is to thread ssDNA electrophoretically into a membrane protein pore creating a temporary shift in pattern of the ionic current. Recording the pattern of shift determines the sequence of DNA.
This is how it works- two proteins, a zipper and a pore
Whilst all other instruments require the sample to be either bridge amplified or be contained and amplified in tiny droplets of emulsion, nanopore can read through the DNA as is. So this is how it works;
- The technology involves two proteins.
- The first protein is an exonuclease introducing one base at a time of the DNA into a second protein – the pore.
- As the bases pass through the pore, it causes a shift in ionic current and this pattern can be recorded.
- A naturally occurring biological protein produced by Staphylococcus aureus ? –hemolysin is used.
- This produces pores in membranes and the bacterium gains its invasiveness through this protein.
- Oxford nanopore developed a mutant ? –hemolysin closely tethered to the exonuclease and DNA is threaded through it thereby differentiating one base at a time. A concept that closely resembles nuclear pore complexes.
Scalability- the entire human genome in 15 minutes
If we were threading one base at a time into a single pore, it would take more than 70 days to sequence the entire human genome! Therefore the only logical thing to do would be to have multiple nanopores working in parallel. The ‘GridION’ contains 2000 nanopores working to produce 150 mb per hour and the ‘MinION’ contains 512 nanopores. They produce extremely long reads about 50,000 bases in length and the ‘GridION’ can sequence the entire human genome in 15 minutes.
Why is it still not commercially available?
The concept of nanopore sequencing was discussed in early 1990s and it was a while before it took the form of a sequencer. But it is still not commercially available. At present, Oxford Nanopore Technology faces its biggest challenge- an error rate of 4%.
Ironing out the errors
The error rate stems from the size of the pores. Single base pairs which are 0.4 nm apart cannot be distinguished accurately from one another owing to the size of the pore through which the DNA is funneled. Another potential reason for error is the rate at which the DNA is translocated through the pore. This rate needs to be reduced from microseconds per base to milli seconds. Oxford Nanopore are working to reduce the errors to less than 1% before its release.
Will it be worth the wait?
The technology promises not only low cost sequencing and reduced sample prep, but also a low cost instrument and extremely long reads- two features that are unavailable right now amongst sequencers. At present, the projected cost may not include that of analysis, but this it is not estimated be any more than the already available technologies.
In conclusion the technology is promising, and with a substantial limitation yet to be discovered, nanopore sequencing still has the potential to be an extremely cost-effective, fast and accurate sequencing technology.