By Monica Heger
The past year saw enormous progress in the field of nanopore sequencing with advances ranging from the design of protein nanopores to the use of graphene in the solid-state field, improvements in slowing down the rate of DNA translocation through the pore, and the ability to distinguish individual bases and epigenetic modifications in the pore.
The number of groups doing research in the field has been steadily increasing for years. According to Oxford Nanopore, there were more nanopore-related DNA patents filed within the last three years than the previous 23 years combined.
Investment in the technology has also held steady, with the National Human Genome Research Institute awarding more than $10 million to groups working on the technology under its "$1,000 Genome" Advanced Sequencing Technology program in 2010 — more than half the total funding for the program in this round (IS 9/14/2010).
Startups and well-established companies alike also invested heavily in nanopores in 2010, including NobleGen Biosciences, which is developing a sequencer based on arrays of solid-state nanopores and optical detection, as well as Roche and IBM, which are co-developing a nanopore sequencer based on IBM's DNA transistor technology (IS 5/25/2010 and 7/6/2010).
Meanwhile, nanopore veteran Oxford Nanopore has expanded its scope from focusing solely on exonuclease sequencing to partnering with academic groups that are working to develop nanopores based on strand sequencing and also working on those strand-sequencing methods in-house. It has also increased its number of academic partners, and is now collaborating with groups working on protein nanopores as well as those working on solid-state nanopores.
While a nanopore sequencing device has yet to be realized, there's a high level of interest because such a device has the potential to offer a completely different type of sequencer than those currently available, said Spike Willcocks, Oxford Nanopore's vice president of business and corporate development.
With nanopore sequencing, DNA does not need to be treated before sequencing, reducing the cost, time, and errors inherent in any sample-prep step. Nanopore sequencing would also require much less starting DNA, and because of its small size and likely simple design, many think a nanopore sequencer offers the best opportunity to really achieve the $1,000 genome.
"The type of products that can be made with nanopores are very different and can give that revolutionary aspect [to genome sequencing] that PacBio or Ion Torrent can't," he told In Sequence.
MspA vs Alpha-hemolysin
This past year saw major developments on two different types of protein pores: alpha-hemolysin, which has been the primary protein researchers have been studying for nanopore sequencing; and newcomer Mycobacterium smegmatis porin A, or MspA, which may offer superior properties than alpha-hemolysin.
"The jury's still out on which pore" is better, Willcocks said.
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On the alpha-hemolysin side, the University of Oxford's Hagan Bayley, also a co-founder of Oxford Nanopore, which supports much of his work, has been leading the charge on engineering an alpha-hemolysin pore optimal for nanopore sequencing, and this year made several advances relevant for strand-sequencing.
This past summer, he demonstrated how to modify the pore to achieve the most robust signal possible, and then in the fall showed that his engineered alpha-hemolysin pore could not only distinguish between the four bases, but could also identify epigenetic changes to the DNA (IS 10/12/2010).
Meanwhile, a group from the University of Washington showed in a proof-of-principle study published during the summer that MspA may in fact offer superior properties (IS 8/24/2010).
Jens Gundlach of the University of Washington, who led that research, said he thought the MspA pore would be ideal because it has just one site within the pore where bases are read, while the alpha-hemolysin pore has three. While Bayley's team has demonstrated that the alpha-hemolysin pore can be engineered so that signal is only read from one of those sites, Gundlach's team was able to demonstrate a lower signal-to-noise ratio because of the design of the MspA pore.
The MspA pore is also much smaller than alpha-hemolysin, enabling only a single base to be contained within the pore at one time, so the signal elicited will only be from that base. In Gundlach's proof-of-principle study, he demonstrated four distinct current signals.
Graphene: the Wonder Material?
Graphene, which has been touted as a wonder material and drawn interest from scientists across many different disciplines, entered the nanopore field this year. Groups from Harvard University, the Kavli Institute of Nanoscience in the Netherlands, and the University of Pennsylvania all demonstrated DNA translocation through graphene-based nanopores during the year (IS 7/20/2010 and 8/3/2010).
The interest in graphene is two-fold. The material is only one atom thick, making it ideal for a nanopore, since that thickness would ensure that only one base is being read at a time. Also, the electronic properties of graphene could enable it to act as both the pore and also the electrodes from which the electronic signal of the bases are read. One problem with solid-state nanopores has been the need to couple a pore with a reading mechanism, but the use of graphene as both the pore and the device from which the bases are read could solve that problem.
Despite the advances in graphene research, much is unknown about the pores. For instance, despite being only one atom thick, ions tend to coat the surface of the graphene, and it is not clear whether those ions add to the thickness of the membrane or not.
Additionally, solid-state nanopores are not as reproducible as protein nanopores, so any slight differences could affect the function of the pore and how the bases are read.
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Slowing Down Translocation
Aside from designing a pore that is both reproducible and demonstrates single-base recognition, the second major hurdle researchers have to overcome is slowing down the rate of translocation enough so that each base can be read as DNA moves through the pore. Right now, even though groups have demonstrated that they can distinguish between the bases in some pores, and have also demonstrated that when voltage is applied to the pore DNA will pass through it, no one has yet to read bases from a moving piece of DNA, because it currently moves too quickly.
However, during 2010 significant advances were made in slowing down translocation enough to be able to read the bases.
Mark Akeson's group at the University of California, Santa Cruz, attached a polymerase to the edge of an alpha-hemolysin pore and showed that they were able to slow down translocation of a single strand of DNA so that each base would be held in the pore for 17 milliseconds, long enough to read the base (IS 9/28/2010).
The method still had problems, though. Namely, the enzyme would dissociate from the pore after only reading several bases. However, in a follow-up study, the team demonstrated that a different enzyme would not dissociate, even after ratcheting through just over 100 bases.
The next steps are to demonstrate that even longer pieces of DNA can be translocated through the pore and also to couple that with reading the actual bases — either through an alpha-hemolysin pore or an MspA pore. Akeson has said that either pore would work with his method.
Nanopores in 2011
The explosion of research in nanopores, particularly over the last year, could lead to some important advances over the next year as advances in pore design, DNA translocation, and base reading are all combined.
Oxford Nanopore's Willcocks said it is reasonable to predict that within the next six months Akeson's method for moving DNA through a pore would be combined with either Bayley's detection method for the alpha-hemolysin pore, or Gundlach's MspA pore.
"There's no reason it can't be done in the next six months," he said.
Additionally, Willcocks said it would be important for someone to demonstrate that long fragments of DNA — one to five kilobases long, for instance — can move through a nanopore. "That will really give credibility to nanopore sequencing."
Akeson too, said reading sequence through a nanopore was imminent. "It wouldn't surprise me if people were reading sequence through a nanopore by the end of the year," Akeson told In Sequence. "It's not a prediction, but it wouldn't surprise me."
Researchers also expect that over the next year more advances will be made in the development of graphene nanopores as well as hybrid nanopores — the combination of a protein nanopore in a solid-state membrane.
This past year, Cees Dekker's group at the Kavli Institute of Nanoscience in the Netherlands created a hybrid nanopore (IS 12/21/2010). The group inserted an alpha-hemolysin pore into a silicon nitride membrane, attempting to combine the reproducibility and specificity of the protein nanopore with the sturdiness of the solid-state membrane.
David Deamer, a pioneer in the protein nanopore field at the University of California, Santa Cruz, said he thought that this hybrid approach could be the future of nanopore sequencing.
"Looking ahead, I would really hope that [the hybrid pore] is going to lead the way into a very stable alpha-hemolysin pore, or maybe some other protein like the MspA pore, where we get great reliability coupled to great stability," he said.
And finally, with graphene nanopores, Willcocks said that the next step in that field would be to show the measurement of a single base within the pore on a strand of static DNA.
Even though the protein nanopore field is more advanced, the solid-state field "has been coming along faster in the last 12 months and there's no reason to think that it won't continue, especially with graphene," Willcocks said.
He cited other groups that would likely make headway in the solid-state field in the coming year, including IBM, which is developing a DNA transistor, and also groups that have been working on DNA tunneling, such as Stuart Lindsay's group from Arizona State University (IS 2/16/2010).
Nanopore sequencing has come a long way over the last year and while many are excited about the prospects and the potential of nanopore sequencing, experts are still reluctant to predict when an actual device will be available for use, even as a prototype, illustrating just how early-stage much of the research still is.
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