Monday, 30 January 2012

Nanopore sequencing: is the hype about to end?

A follow-up post to this one explains what nanopores are and how they can be used for DNA sequencing.

There has been a lot of hype around Nanopore sequencing for a number of years. The promise of very long single molecule sequencing with nucleotide modifications being directly read out is the holy grail of sequencing technology. The fact is that it has been hard to translate a relatively simple concept into reality.

For the past three years attendees at AGBT have been waiting for Oxford Nanopore to speak. To date they have been very reluctant to say much publicly and although it has not been confirmed, the rumour mill is hot with speculation that this year is finally the year that they will talk.

But what will they talk about and is the Next Next-Gen (N2GS) just around the corner?

What am I hoping to hear about: I am hoping for a genome, PhiX would be OK (any nanopore sequence would be better than nothing), but I’d admit to being disappointed if this was all. It would be great to see a complex genome presented, Yeast or C. elegans. Of course what I really hope is that they will talk about their sequencing of a Human genome and if I allow my imagination to run away then I am looking forward to details of a long-read single molecule 1000 genome project.

What do Oxford Nanopore offer: ONT are developing nanopore-based technologies, the most interesting of which for this post is DNA sequencing.

ONT’s sequencing that has been discussed publicly uses an α-haemolysin pore coupled with an exonuclease. Rather than feeding an intact DNA strand through the pore and reading out the bases, the exonuclease-sequencing approach cleaves each base from the end of the DNA strand, those bases translocate through the pore and are detected and the DNA seqeunce is read out. Many thousands of these nanopores are required to run in parallel to sequence a genome. And part of the parellelisation comes in the form of the GridION system, where multiple sequencing chips can be run together.

Strand-sequencing has not been dropped by ONT though and Hagan Bayley has modified the pore to improve discrimination of bases. Others have also demonstrated methods to slow down the translocation of DNA through the pore by coupling it to a polymerase, which ‘ratchets’ the samples through a base at a time.

The GridION system was ‘reviewed’ in a post over at Genomes Unzipped by Luke Jostins almost exactly one year ago. And whilst this post has scant details on the sequencing to be fair very little additional has been revealed in the following 12 months. Lukes post does describe the compute-cluster-like architecture that ONT have developed to house future sequencers. A user could buy one or one thousand and do as much or as little sequencing as they need. What the impact will be on service providers like BGI will be interesting to see.

“Run until” technology: ONT’s exonuclease or strand-sequencing approaches, packaged in the GridION format, will allow scientists to load the instrument and continue to sequence until ‘enough’ data has been generated. How much is enough is determined by the user ahead of the run and the real-time analysis and monitoring will mean only the required data are generated.

It is theoretically possible to make use of this approach, or something similar today. By running highly multiplexed samples and performing analysis between runs it would be possible to generate almost exactly the required depth of sequencing per sample. Once a sample has been shown to be of high quality then only the number of indexed reads is required to calculate how much more sequencing is needed. This could be implemented on the other sequencing platforms, but I suspect is too difficult to implement in most labs today. Even if the efficiency gains could be worthwhile.

ONT and Illumina: In 2009 Illumina invested $18M in ONT and bought an exclusive license to "market, sell, distribute, and service BASE( BAyley SEquencing) technology.What this means for ONT and Illumina today is less clear and I am not certain if other milestone payments have been paid. Illumina certainly want to stay ahead of the competition (LifeTech, Complete and Roche). ONT might be their not-so-secret weapon.

What does the future hold: Dr Sanghera (ONT CEO) was quoted saying that the $1,000 genome will be possible “within three to five years” in a March2011 interview with The Economist. Things have sped up faster than ever in the last year and the $1000 genome looks like it is already here. Can ONT deliver us the $100 or $10 genome? I am certainly looking forward to having them piling the heat on Illumina and Life Tech.

Lastly, back in 2009 when Clive Brown ONT CTO) was interviewed in BioIT World he said "before launching a product, you have to run it in house for months, doing genome-centre type things". Hopefully we are about to find out exactly what those genome-centre type things are.


  1. ONT has said they will be announcing something right around the time of AGBT. Whether it is at AGBT or not, I can't wait to hear what they have for us!

  2. I don't doubt that they will announce some form of sequencing at/around AGBT, I would be most interested in hearing how they can scale up the multiplexing. There are no solid-state technologies capable of doing that today, and it is difficult to see how you radically scale up and address biological pores in the millions+ range.

  3. I'd expect them to use the GridION as the multiplexing platform with multiple samples per 'node'. I guess the cost of a chip is going to be a significant factor here as a "run until" process that only requires a few thousand reads will process lots of chips. Perhaps an end labelling with an index would work as well? However it would require sample prep and that is something many users are hoping will dissapear.

  4. That is the "obvious" answer, but... you touch upon the core problem - 1M reads is A LOT of protein pores and A LOT of chips. The signal quality of a protein pore will probably high, but the ability to scale as needed will be nil. At least this is what I believe.

  5. I expect many people in the AGBT audience to be doing some quick maths during Clives talk ready to ask exactly those kind of questions. It would also not surprise me if ONT suffer from the same loading issues as other platforms so 1M pores might only generate 300,000 reads in the first release anyway.

  6. I know only that it has been hard to translate a relatively simple concept into reality.