James Clarke, Hai-Chen Wu, Lakmal Jayasinghe, Alpesh Patel, Stuart Reid, Hagan Bayley (2009). Continuous base identification for single-molecule nanopore DNA sequencing Nature Nanotechnology DOI: 10.1038/nnano.2009.12
The clever boys and girls at Oxford Nanopore Technologies – one of the most quietly impressive contenders in the hotly-contested next-generation DNA sequencing race – have a new paper out in Nature Nanotechnology today. The paper demonstrates proof of principle for a crucial step in their approach to DNA sequencing, the accurate recognition of DNA bases as they pass through a tiny protein nanopore.
Oxford’s approach is outlined in cartoon format in this video:
(See the Oxford Nanopore website for a better-quality Flash version of the video.)
Put simply, the system works by sequentially chewing DNA bases off the end of a long strand and then detecting each cleaved base as it falls through a protein nanopore. In today’s paper the company demonstrates the use of engineered nanopores to achieve accurate recognition of five different DNA bases (the standard A, C, G and T, as well as methylated C).
Here’s the crucial figure:
The picture above shows the traces left by the four different DNA bases due to the different effects these molecules have on electrical current flowing across the nanopore. Although there’s a fair bit of noise at the molecular level, these signals provide surprisingly accurate base detection: the histogram on the left shows the fraction of bases correctly assigned (base-calling errors fall in the troughs between the four peaks). The average accuracy is 99.8%, and importantly the errors aren’t random: where there is ambiguity, there are only two possible states (rather than four). That type of error constraint will make it much easier for downstream analyses to minimise the impact of base-calling errors.
There’s still plenty of work to be done before this technology can be converted into a commercial platform, but I understand that the company has made substantial progress since the work described in the paper was completed over six months ago. Clearly Oxford is doing something right – the recent $18 million cash injection from sequencing giant Illumina, with a promise of more funding to come if Oxford met specific (unnamed) technical benchmarks, was an impressive vote of confidence.
Oxford’s technology strikes me as the most exciting in the emerging third-gen arena: the nanopore approach offers some hefty potential advantages over competing technologies.
Firstly, the platform detects DNA directly rather than relying on
indirect labelling steps, which is important for several reasons: it
means that sample preparation should be trivial (essentially just DNA fragmentation), reagents will be much cheaper, and there will be no need for amplification of the input DNA – eliminating a major potential source of bias.
Secondly, this paper demonstrates that the system can detect the modified DNA base methyl-cytosine directly,
which will be extremely useful for epigenetics (the analysis of
physical modifications of DNA, which can have effects on the levels of
gene expression). Current systems for detecting methyl-cytosine require
a fiddly bisulphite substitution step; direct reading of modified bases
would be a huge advantage.
Thirdly, if the company can combine their nanopore reader with a DNA-cleaving enzyme with sufficiently high processivity, the platform would potentially offer extremely long read lengths.
The ability to sequence single molecules of DNA with high accuracy over
thousands of base pairs would make assembling whole genomes much easier
and far more accurate than is the case for current platforms (which
have read lengths ranging from around 35 to 500 bases).
Finally, the simplicity of the system’s readout as electrical signal means that downstream data processing and storage are easier, and that the system is readily scaleable – manufacturing techniques for handling high-density electrical sensors are already very well-established. That means creating a massively parallel array of nanopores to sequence huge numbers of DNA molecules simultaneously should be relatively straightforward.
Oxford will be up against a strong rival in Pacific BioSciences, who look set to launch their own commercial long-read single-molecule sequencing platform in 2010, but the advantages of nanopore technology and their backing from Illumina make the UK-based company a serious contender. I’m looking forward to seeing how this field evolves in 2009.
Media coverage: So far, a nice piece by Kevin Davies in Bio-IT World and a story in the Times focusing on the broader implications of cheap sequencing technology. A new blog, Biotechnically Speaking, focuses on the implications of this technology for epigenetic research.