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Targeting One Megabase Per Second, VisiGen Plans to Offer Sequencing Services by 2009

VisiGen Biotechnologies has great ambitions: The company is not only targeting the $1,000 genome, but the $1,000 genome in less than a day.
The Houston-based company, which received an undisclosed equity investment from Applied Biosystems in late 2005, was founded in 2000 to develop a real-time single-molecule sequencing-by-synthesis technology that will eventually read one million bases of DNA per second. It hopes to offer a sequencing service based on its technology by the end of 2009, followed by an instrument release about two years after that.
“With that kind of throughput, you can get 10X coverage of a human genome in way less than a day,” said Susan Hardin, VisiGen’s founder, president, and CEO.
Last week, the US Patent and Trademark Office granted VisiGen a patent, the first of approximately 30 patent applications the company has filed on various aspects of its technology. The patent, No. 7,211,414, is entitled “Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity.”
The company also licenses a technology from the National Institutes of Health (see Short Reads in this issue).
VisiGen’s approach relies on monitoring DNA polymerase, which the company calls a “nano-sequencing machine,” as it synthesizes DNA. The polymerase is labeled with a donor fluorophore and incorporates nucleotides labeled with acceptor fluorophores into a DNA strand. Whenever a nucleotide binds to the active site of the enzyme, energy is transferred from donor to acceptor in a process called fluorescence resonance energy transfer, or FRET. The acceptor gives off light of a particular wavelength that is detected and identified by VisiGen’s system.
By tracking many polymerase molecules in parallel as they synthesize DNA, VisiGen hopes to achieve a throughput of a million bases per second. Read lengths will reach about 1,000 base pairs, limited by how long the polymerase stays on the DNA, and how long the polymerase label will stay intact.
The system is able to track each polymerase in real time because the enzyme completely removes the acceptor label before it binds to the next nucleotide. Removing the label, which is attached to the last, or gamma, phosphate of the nucleotide, also results in “absolutely natural DNA” with no modification that could slow down the polymerase, Hardin said.
Measuring signals from single molecules requires the use of “very sensitive cameras,” she said, but would not offer any further details. Camera technology also limits how tightly the polymerases can be packed in a single field of view. Depending on how quickly camera technology advances, it could be “quite reasonable to get to 100,000 [polymerases], and a million is probably towards the upper limit,” she said.
After monitoring many polymerases for a certain amount of time, the camera moves on to an adjacent field of view and watches another batch of enzymes as they make DNA.
Currently, the “preferred strategy” is to randomly immobilize the polymerases on a surface, which VisiGen has optimized to reduce background noise from labeled nucleotides. “That keeps the polymerase in the illumination volume at a particular position, so that we get a more consistent signal,” Hardin said. Also, it helps to keep the distance between donor and acceptor fluorophore fairly constant, she added.
So far, company researchers have been working with reactions containing two different acceptor labels. In order to be able to sequence DNA, they have to increase that number to four. More labels with different signal strengths in the reaction mix, however, create a challenge that is “certainly not insurmountable, but it requires very careful engineering of the detectors,” Hardin said.
Right now, the scientists are focusing on “making sure we understand what every single signal means in our system,” Hardin said.
“We are following approximately 50 different attributes that are associated with the FRET information that’s detected by the system, discerning noise from an incorporation event, versus a non-productive binding, versus a misincorporation event, versus a collisional interaction. Every one of those types of signals has different attributes associated with them, and we are working to assign confidence values to every single signal that shows up in our system.”
Keeping the noise down might indeed be one of the major challenges. “I think there is no fundamental flaw in this method, I think it can work,” said Ido Braslavsky, an assistant professor in the department of physics and astronomy at Ohio University in Athens. “The problem might be that the signal-to-noise ratio for measuring a single molecule in real time can cause a high error rate,” he said. “But this can be alleviated by throughput. So there is a trade-off between error rate and throughput.”
As a postdoc in Steve Quake’s lab at Caltech, Braslavsky published a paper on single-molecule sequencing in the Proceedings of the National Academy of Sciences in 2003 and recently wrote a book chapter on this topic. He also consulted for Helicos BioSciences, which has developed a single-molecule sequencing technology that is based on his work, but does not measure the incorporation of nucleotides in real time.

“With that kind of throughput, you can get 10X coverage of a human genome in way less than a day.”

Recently, Helicos said the first version of its sequencing instrument, which it plans to commercialize by the end of this year, will have a throughput of 25 million to 90 million bases per hour, and that technical improvements could increase the throughput to 1 billion bases per hour, translating to about 280,000 bases per second.
VisiGen plans to provide sequencing services “towards probably the end of 2009,” Hardin said, with an initial throughput that will likely be lower than one million bases per second. Around the same time, it plans to start beta-testing the instrument. Likely beta-testers will be SeqWright, the sequencing service provider that shares a building with VisiGen and invested an undisclosed amount of cash in the company in 2004 and 2005, and Richard Gibbs’ group at the Human Genome Sequencing Center at Baylor College of Medicine. Gibbs, director of the HGSC and a founder of SeqWright, sits on VisiGen’s board of directors.
Short term, the company plans to commercialize a labeling reagent that is currently in beta-testing with an undisclosed party: an ATP molecule that is fluorescently labeled at its gamma phosphate and could replace radioactive labeling reagents in applications such as labeling of DNA or proteins. However, that product is “not the high-priority effort within the company,” Hardin said.
Hardin founded VisiGen in May of 2000 along with four colleagues from the University of Houston: Xiaolian Gao, a professor of biology, biochemistry and chemistry; James Briggs, an associate professor of biochemistry; David Tu, a professor of biology and biochemistry; and Richard Willson, a professor of chemical engineering.
Besides the investments from SeqWright and ABI, the company has been funded by several government grants and contracts, worth more than $8 million in total, from the Defense Advanced Research Projects Agency, the National Human Genome Research Institute, and the National Institute of General Medical Sciences.
Hardin, the only company founder who works full-time at VisiGen, would not disclose the total number of employees but said the company funds 12 people through its NIH grants.
Last fall, VisiGen entered the Archon X Prize for Genomics competition, which is promising $10 million to the first team to sequence 100 human genomes in 10 days for no more than $10,000 per genome (see GenomeWeb News, In Sequence’s sister publication, 10/23/2006).
But Hardin is not only interested in providing technology for sequencing the human genome quickly and cheaply. “We need to make sure that physicians are going to be able to deal with this kind of information, and we need to make sure that we have controls in place,” she said. “I strongly believe that it needs to become, relatively soon, an active part within our company.”

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