Correction to this article

IT WAS one of the largest international scientific collaborations ever undertaken and it led, eventually, to a landmark accomplishment. Yet even with a budget of more than $3 billion and the participation of thousands of scientists, the process of mapping the human genome still took more than a decade. The technology has improved enormously since then: it is now possible to sequence a human genome in about eight days, at a cost of around $10,000. But researchers dream of being able to complete the process in a matter of hours, or even minutes, for less than $1,000. Genome sequencing could then become routine, making it much easier to identify the genetic basis of diseases or isolate the factors that cause a drug to work in one patient but not another.

The most promising way to make such cheap, rapid sequencing a reality is an approach called “nanopore sequencing”. This involves drawing individual strands of DNA through tiny nanoscopic holes, or pores. The idea is that individual base pairs (the four chemical “letters” of the DNA alphabet) can then be read off one at a time as they pass through the nanopore. “My own vision is a 15-minute genome,” says Hagan Bayley, a chemist at the University of Oxford who is a pioneer in the field of nanopores. “While you are being diagnosed at the hospital your entire genome would be sequenced.”

Sequencing technology has come a long way since the Human Genome Project, but it still requires DNA to be copied millions of times, a step called amplification, and labelled with fluorescent tags. It is a time-consuming process and a significant bottleneck, says Gordon Sanghera, the chief executive of Oxford Nanopore, a firm set up in 2005 to commercialise Dr Bayley's work on nanopores.

“Today all the systems that are out there have to chemically label the DNA and copy it so they have enough to read it,” says Dr Sanghera. “So the current state of the art spends between five and ten days just preparing the DNA.” With nanopores, however, virtually no chemical pre-processing is required and no amplification. Instead a single strand of DNA can be read, one base pair at a time, and without the need for labelling. Moreover, existing sequencing techniques involve breaking DNA into small chunks of less than 100 base pairs. These chunks have to be sequenced many times to find identifiable overlaps so that they can be pieced together, says Gustavo Stolovitzky, the head of the functional genomics and systems biology group at IBM, a computer giant. Nanopore sequencing can cope with much longer strands, which should help speed things up.

The hole story

Coaxing individual strands of DNA, some 50,000 times thinner than a human hair, through holes just a couple of nanometres wide (a nanometre is a thousand millionth of a metre) may sound impossible. But something similar happens in nature all the time, in the form of pore-forming proteins, called ion channels, that punch holes in the membranes of cells and selectively allow ions to pass through them. Many biological processes depend on the ability of ion channels to regulate the flow of ions through cellular membranes.

In the 1990s, inspired by the elegance of these proteins, Dr Bayley started looking at how ion channels could be used as biomolecular sensors. He started off with a lipid bilayer—a very thin biological membrane with a high electrical resistance. An ion channel is used to create a tiny hole in this layer, and a molecule with a slight negative charge is placed on one side of it. Applying a voltage across the bilayer causes the molecule to push its way through the pore to the other side. Crucially, as it passes through the pore, the molecule produces characteristic disruptions to the flow of current which can be used to identify what sort of molecule it is.

“Nanopore sequencing is gaining momentum, with new developments occurring almost every month.”

By 1996 a team of researchers from Harvard University and the University of California, Santa Cruz, including Daniel Branton, David Deamer and John Kasianowicz, had found that using one protein in particular, alpha hemolysin (AHL), it was possible to get single strands of DNA, not just small molecules, to pass through a nanopore. This, they realised, might lead to a new way to sequence DNA. But threading a strand through an AHL pore and detecting the individual bases at the same time is very difficult. So Oxford Nanopore has also been looking at an alternative approach, called exonuclease sequencing, in conjunction with Illumina, an American firm that is the market leader in rapid sequencing. This also involves a nanopore in an AHL protein, but the DNA strand does not pass through the nanopore intact. Instead an enzyme, called an exonuclease, is attached to the top of the AHL. It cleaves individual base pairs from the DNA strand and feeds them through the pore to be detected one at a time.

Dr Sanghera says Oxford Nanopore has developed an electronic cartridge system called GridION, based on “lab on a chip” technology, for exonuclease sequencing. Each cartridge contains multiple nanopores (though the firm will not say how many) and all the microfluidics and electronics to carry out the preparation, detection and analysis. These are plugged into a rack-like device that resembles a computer server. And, like servers, the racks can be combined in vast cabinets to carry out large-scale analysis, sequencing DNA or simply detecting small molecules or proteins. Dr Sanghera says this system is almost ready for commercial launch.

Even so, the ultimate goal is to perfect single-strand DNA sequencing, threading large sequences of DNA through nanopores and reading them as they pass through. This would be faster than exonuclease sequencing. It would also be more accurate: because the bases are still attached to one another, there is no chance that they will pass through the nanopore in the wrong order. One of the reasons single-strand sequencing is so difficult, however, is that the combined thickness of the protein and bilayer means that as many as 15 bases may be inside the nanopore at any one time. In addition, the speed at which the strand passes through must be carefully controlled to allow enough time to detect each of the bases.

The ratchet effect

In late 2010 Dr Bayley found a way to address the first of these problems, and Mark Akeson at the University of California, Santa Cruz, found a way to tackle the second. By modifying the AHL protein to create a constriction within the nanopore, Dr Bayley showed that it is possible to ensure that the variation in current is determined by a single base, which can then be identified. Dr Akeson, meanwhile, created a clever ratchet arrangement by attaching a polymerase enzyme to the AHL protein. This can ensure that the strand passes through the pore at a rate of one base every 20 milliseconds, which should be slow enough to detect each one.

Yet even a nanopore reading 50 bases per second would take about two years to sequence a whole genome, which contains around 3 billion genetic letters. So it will also be necessary to find ways to get lots of nanopores to work in parallel, says Dr Bayley, with each sequencing different sections of a genome simultaneously.

An alternative approach, which has been gaining ground in the past couple of years, is to dispense with proteins and create artificial nanopores in solid-state materials. IBM and 454 Life Sciences, a division of Roche, a drugs giant, have been working together to this end, using an electron microscope to punch three-nanometre holes in membranes that are ten nanometres thick. These membranes are made of three layers of a conducting material, titanium nitride, separated by insulating layers of silica. When a voltage is applied across the membrane, a strand of DNA is drawn into the pore. As it enters, a separate electric field is applied across the metal layers of the membrane, trapping the first base pair. By flipping this field's polarity the DNA strand can be ratcheted along, one base pair at a time.

This can be done very quickly, says IBM's Stanislav Polonsky, co-inventor of the technology. In order to get a strong enough signal to read the bases it is only necessary to trap each one for around a millisecond, he says. Most of this work has been carried out in computer simulation, but the ratcheting mechanism has been demonstrated in the laboratory. Moreover, it can move the DNA in both directions, which would have the huge benefit of allowing error correction to be carried out, says Stephen Rossnagel of IBM. If solid-state nanopores can be made to work, existing chipmaking technology could then be used to create massively parallel devices for well below $100 a chip, he says.

Others are also looking at solid-state nanopores, including Jene Golovchenko at Harvard University, with whom Oxford Nanopore is also collaborating, and NabSys, a spin-out from Brown University in Rhode Island. A variation of this approach uses nanopores in graphene, a sheet-like form of carbon that is one atom thick. Because such membranes are so thin, only a single base will be present within the pore at any one time, making detection easier.

According to preliminary experiments by one group, headed by Joshua Edel at Imperial College London, this approach should be able to sequence an entire genome in just a few minutes. Another group, led by Marija Drndic at the University of Pennsylvania, who made the first graphene nanopores in 2008, has found that adding a layer of titanium oxide a few molecules thick to the graphene membrane lets the DNA pass through more easily and reduces the noise level when measuring the current, which should improve accuracy. Dr Bayley is looking into solid-state pores too, and is also exploring a hybrid approach that combines protein-channel nanopores with solid-state membranes.

There is a clear sense that the field of nanopore sequencing is gaining momentum, with new developments occurring almost every month. Dr Sanghera believes that the $1,000 genome will be possible “within three to five years”. The benefits could be vast, he says. Cheap and rapid whole-genome sequencing will, for example, make it possible to sequence the entire genomes of cancerous and healthy cells in individual patients to see what has changed. It should become easier to determine which drugs will work best in a particular patient, and why. It will also be possible to construct a fine-grained picture of human evolution, and of how and when humanity spread around the world. Nanopores are tiny, but if they can be harnessed, the consequences could be momentous.





Correction: An earlier version of this article wrongly attributed the initial threading of DNA through nanopores made using AHL proteins, in 1996, to Hagan Bayley. This was corrected on March 11th 2011.