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By Julia Boguslavsky

 November 15, 2003 | When scientists at 454 Life Sciences set out to build a better, faster, and cheaper DNA sequencer, they quickly realized that the sequencing step is only one piece of the puzzle.

"In order to sequence a whole genome, researchers need not only a fast sequencer, but also improved sample preparation and amplification, as well as simplified genome assembly," says 454's president and CEO, Richard Begley. "In order for this technology to propagate, the whole instrument has to be sufficiently small and inexpensive to be adopted by clinics. We had to miniaturize and simplify each step."

Relying on advances in nanotechnology, scientists at 454, a subsidiary of CuraGen based in Branford, Conn., developed an integrated end-to-end instrument that performs hundreds of thousands of reactions in parallel — from sample preparation, amplification, and sequencing to data storage and bioinformatics. The system allows whole-genome analysis within days, using a single operator.

"We are not just working on a fast sequencer, but a personalized genome center," Begley explains. "We can now fragment and amplify a virus and prepare it for sequencing in a volume the size of my thumb. And all this can be done by one person. It's a drastic simplification."

Sequencing in parallel: The PicoTiterPlate contains 300,000 etched wells, which house individual sequencing reactions. The 44-micrometer wells are imaged using a scanning electron microscope.
The sample preparation step involves fragmenting the genome, isolating the fragments, and binding them to beads for solid-phase DNA amplification. The entire process takes 8 to 12 hours and does not require colony picking and microplate handling.

For sequencing, the DNA-carrying beads are inserted into PicoTiterPlates. The smallest available plate, the size of a microscope slide, contains 300,000 75-picoliter wells. (To accommodate larger genomes, the PicoTiterPlates also come in 800,000-, 1,200,000-, and 2,400,000-well formats.) The beads are just slightly smaller than the wells, ensuring that only one DNA-carrying bead is inserted into each well.

The sequencing-by-synthesis reaction on each fragment occurs simultaneously in all wells. The reaction generates unique light signals, which are detected by a CCD camera via fiber optics at the bottom of the plate. This enables 454 scientists to sequence 100-base-long fragments in each well, which are then reassembled into the whole genome sequence.

"One operator can go through sample preparation and amplification, sequencing, and assembly — all in the space of an office," Begley says. "The premise of the program is to miniaturize every step of the sequencing process and run it massively in parallel in order to shorten the sequencing time. As we move on to bigger genomes, we will continue the process in parallel simply by adding more wells to the PicoTiterPlates and clustering multiple instruments for simultaneous operation."


From Medicine to Biodefense
454 says it is now able to sequence a virus genome in 24 hours (see "From Sanger to 'Sequenator,'" Oct. 2003 Bio·IT World, page 1). And at the recent Genome Sequencing and Analysis Conference, Kent Lohman, 454's senior director of molecular sciences, presented the first attempt to sequence a bacteria. The entire process took five days and yielded 94-percent coverage of Staphylococcus aureus. "Once we optimize this process, we should be able to sequence a bacteria in a day — by Christmas," Begley says. The current throughput for each instrument running the smallest PicoTiterPlate approaches 2.5 megabases per hour.

Others to Watch

Keep an eye on these companies for advances in sequencing and genome analysis technologies:

· OpGen, Madison, Wis. (www.opgen.com)

· ParAllele BioScience, South San Francisco, Calif. (www.p-gene.com)

· Perlegen Sciences, Mountain View, Calif. (www.perlegen.com)

· Solexa Ltd., Little Chesterford, U.K. (www.solexa.com)

· U.S. Genomics, Woburn, Mass. (www.usgenomics.com)

· VisiGen Biotechnologies, Houston, Texas (www.visigenbio.com)
The company plans to systematically scale up to bigger genomes, while improving the cost, throughput, and ease of use of the entire sequencing process. Begley expects to proceed in three "logical phases." The first phase, focused on sequencing viruses and bacteria, should be under way by the end of this month through contract sequencing services. The production instruments and kits are slated to ship by spring of 2004. In addition to standard drug discovery applications, the ability to rapidly and cost-effectively sequence small infectious organisms opens the door to a number of applications, such as epidemiological studies, monitoring bacterial mutation, and engineering better bioremediation bacteria.

The second phase aims at sequencing fungi, small model organisms, and individual human chromosomes, which will start providing useful information about multigenic diseases.

The entire human genome should be attainable at a "reasonable" cost within a few years, Begley says. Whole-genome screening will allow drug development companies to conduct association studies for disease gene identification, select patients for clinical trials, and deliver on the promise of personalized medicine.

Julia Boguslavsky is the conference director for Cambridge Healthtech Institute. She can be reached at julia@thebiotechwriter.com.


PHOTO IMAGE FROM MICROSCOPE OF COURTESY 454 LIFE SCIENCES



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