SCIENCE & TECHNOLOGY

THE $1,000 GENOME New methods aim to drive cost of sequencing an individual human genome to below $1,000 CELIA HENRY ARNAUD, C&EN WASHINGTON

THE HUMAN GENOME PROJECT IS

long done, and the entire genome is sequenced. W e know the order of the adenines, guanines, cytosines, and thymines. End of

story, right? Wrong. It's only the beginning. The challenge nowis to find away to integrate genomic information into health care.

Sequencing is still prohibitively expensive. The Human Genome Project spent billions of dollars to sequence a single genome. The price has fallen significantly since then to about $10 million for a genome the size of a human's. But even that fraction of the origi­nal amount is way too expensive to make genome sequencing practical for individual medical decisions.

But what if it cost only $1,000 to se­quence an individual human genome? Sud­denly, sequencing every person's genome would be within reach.

That's just what could happen if the projects funded by the Revolutionary Ge­nome Sequencing Technologies program succeed. Earlier this year, the National Hu­man Genome Research Institute, part of the National Institutes of Health, awarded nine grants totaling more than $25 million, each with the goal of reducing the cost of sequencing a genome to $1,000 or less.

One grant recipient, Hagan Bayley of the University of Oxford, credits N I H with taking the risk to fund speculative projects. "For a relatively modest amount of money, it brings together several excellent groups to carry out research that is quite a bit riskier

than usual," he says. "In this particular case, the payoff in medicine will be huge."

The sequencing technology from Hous­ton-based VisiGen Biotechnologies is probably the closest to bearing fruit. Su­san Hardin, company chief executive offi­cer, believes that a number of features are needed to drive down the cost of DNA sequencing, including a single-molecule approach, massively parallel arrays, and real-time detection. VisiGen is working to incorporate all three in its technique, which uses the enzyme DNA polymerase and the nucleotides themselves to identify the bases directly as a complementary DNA strand is synthesized.

VisiGen exploits DNA replication by putting a donor fluorophore on the enzyme and a different color acceptor fluorophore on each of the four types of nucleotides. When a nucleotide is incorporated into the growing DNA strand, the attached accep­tor fluorophore gives off light, the color of which identifies the base.

The company is on track to launch a commercial sequencing service based on the technology by the end of 2007, according to Hardin. So far, they've been able to sequence DNA 28 bases at a time. The assembly of the data into longer sequences will become more straightforward as they are able to work with longer DNA strands.

Another project is focusing on microflu-idic handling of the sequencing assay. A team at Duke University, Stanford University, and Advanced Liquid Logic, headed by Duke

FLASHING LIGHTS VisiGen's sequencing technology incorporates a donor fluorophore on the DNA polymerase and a different acceptor fluorophore on each nucleotide.

electrical engineering professor Richard B. Fair, uses voltage control to manipulate water droplets on a hydrophobic surface. The water droplets serve as indrvidualfy addressable re­action vessels for pyrosequencing, a method whereby incorporation of a nucleotide into the growing complementary DNA chain re­sults in the release of pyrophosphate, which goes through an enzyme cascade and gener­ates visible light.

To do the sequencing, the team immobi­lizes the DNA on a substrate and then uses the droplets to present nucleotides to the DNA. By collecting the pyrophosphate in the droplets and doing the detection else­where, the researchers speed up the process by continuing the nucleotide incorporation while the pyrophosphate reactions run to completion somewhere else.

Right now, pyrosequencing is limited to lengths of about 100 bases. Fair and his colleagues are working to push that limit to 350 and more. Once they demonstrate that all the reagents and reaction products are compatible with their voltage-driven meth­od of manipulating the droplets, they plan to do proof-of-principle experiments and then send the technology to the genome-sequencing center at Stanford University to sequence larger pieces of DNA.

SEVERAL OF the projects focus on more speculative technologies such as nano-pores. Nanopores reduce cost by speeding up the process and by eliminating the need for DNA amplification and for expensive reagents such as fluorescent nucleotides. One of the challenges with nanopores is how to differentiate the bases. Each proj­ect is putting its own twist on meeting that challenge.

In one version, biophysicist Aleksei Aksi-mentiev, electrical engineer Gregory Timp, and coworkers at the University of Illinois, Urbana-Champaign, are drilling synthetic inorganic nanopores sized to fit single nucleotides through multilayered silicon structures in which two semiconducting plates are separated by a dielectric layer. DNA passing through the pore induces an electrical signal at the semiconducting plates. The plan is to distinguish the bases by their dipole moments.

The problem is that the DNA passes through the pore very quickly, Aksimen-tiev says. "We somehow have to trap the molecule in the pore," he says.

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Jingyue Ju of Columbia Univer­sity is also using synthetic inorganic nanopores. His detection method relies on the fact that the nucleo­tides block the current flowing through the nanopore. He hopes to make the amplitude and dura­tion of that blockage different for each nucleotide.

To accentuate the differences among the nucleotides Ju will chem­ically modify them. Once the DNA bases are distinguishable, he still faces the challenge of slowing down the movement of the DNA through the nanopore for detection.

Despite the sample prep involved with Ju's method, he thinks it will significantly hasten DNA sequenc­ing. "The nanopores provide a sim­ple method to detect DNA at the single-molecule level without in­troducing any separation steps," he says. Ju believes that it will be pos­sible to detect DNA with single-base-pair resolution with nanopores within five years. Distinguishing repeating stretches of DNA will take longer.

Not all of the nanopore projects are based on synthetic nanopores. Oxford's Bayley and Reza Ghadiri at Scripps Research In­stitute are developing protein nanopores for DNA sequencing based on the protein hemolysin. The protein is embedded in a membrane, and the current that flows through the pore changes as a base passes through.

Just as with synthetic nano­pores, hemolysin-based nano­pores must be tweaked to slow down the DNA. "We want to reach some compromise between it just speeding through like a blur, like a car shooting by that you can't identify, and it just sit­ting there,,, Bayley says. ^ |

They want to place a con­striction or recognition site in g ^ the pore to help slow down the DNA. "That recognition {site} might be as simple as a physical constriction on the pore," Bayley says, "or it might be as sophisti­cated as attaching a few bases to the pore so that the DNA sticks and hops along as it moves through." They also are thinking about using enzymes to thread the DNA through at a controlled rate or increasing the viscosity inside the pore.

Bayley imagines using thou­sands of pores in parallel, probably

PINHOLE Aksimentiev and coworkers plan to sequence DNA with a nanopore constructed by drilling a hole in a multilayered silicon device. Green (poly) = polycrystalline silicon; yellow = oxide surface.

sequencing at the rate of a base per millisec­ond. The impact will be even greater if they can sequence lengths of DNA of thousands or even hundreds of thousands of bases.

While other researchers are pushing in­novation, Bhubaneswar (Bud) Mishra's goal is to avoid novelty as much as possible. The New York University computer scientist's goal is "to keep it as simple as possible.,, He approaches the challenge as an engineering project rather than novel science. "I don't

THREADING DNA DNA, represented here as a string of beads, moves through the opening of a hemolysin nanopore embedded in a lipid bilayer. The pore contains a constriction with a recognition element represented by the light green ring. Each nucleotide causes a different amount of current to flow through the pore, as indicated by the different amplitudes in the chart.

want to use any phenomenon that's not well-understood or well-charac­terized," he says. 'The idea is to build on things that are true and tried."

Mishra is combining existing technologies and using calculations to see just how accurate they have to be to drive the cost down. Af­ter considering a number of tech­niques, Mishra's team decided to use the methods of optical map­ping and sequencing by hybrid­ization because they seemed most likely to be most cost-effective. In optical mapping, individual DNA molecules are stretched on a sur-

£ face, cut with restriction enzymes, 3 labeled with fluorophores, and im-° aged. In sequencing by hybridiza-| tion, probes with known sequences § that are six to eight nucleotides

long hybridize to the DNA being sequenced, thereby revealing the sequences that are in the DNA.

Optical mapping places markers along the genome, thus breaking the problem into manageable chunks that can then be sequenced by hybridization.

Experimentation is combined with sta­tistics to solve what is essentially an opti­mization problem: What combination of parameters will yield the least number of experiments, and therefore the lowest cost, to get an accurate sequence?

The more replicates one can | run per genome, the greater I the certainty that the sequence i determined is accurate. If the £ required number of replicates | is too large—say, 1 billion— s then the technique is not useful,

Mishra says. What Mishra's team discov­

ered in combining the tech­niques is that they obey what Mishra calls a "computational phase transition." If the parame­ters are below a certain level, the probability of getting an accurate sequence is zero, but if they are above that level, the probability jumps to one. "Whether anyone can make a correct map or not very sensitively depends on those parameter values," he says. "It's all or nothing."

Only time will tell whether any or all of these methods will deliver the $1,000 genome. The indications are that they should work. As Bayley says, "There's nothing fundamentally against the laws of physics here." •

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