SEQUENCING · Conference control passes from TIGR to the Venter Institute, but sequencing technology marches on.

By John Russell

November 19, 2004 | In its heyday, the Genome Sequencing and Analysis Conference (GSAC) attracted thousands of researchers hungry for information about the latest breakthroughs in sequencing technology. At the 16th GSAC, held in September in Washington, D.C., The Institute for Genomic Research (TIGR) founder and current chairman J. Craig Venter raised some eyebrows by stating that attendance had been deliberately limited to 500.

Barely a week later, there was confusion about whether the conference was being shut down when GenomeWeb reported that conference organizer TIGR notified vendors by e-mail that there would be no 17th GSAC. "The story that came out was not one that anybody knew was coming out and didn't represent what's actually happening," Venter told Bio·IT World. (See "GSAC Returns to Its Roots")

"Some of the fault was with the internal team, with the confusion at TIGR," said Venter. What is changing is that conference management is moving from TIGR to the Venter Institute, and the conference dates will be pushed ahead. "I've chaired it for 16 years and I'll be chairing it next year," said Venter. "We may modify the name going forward, but GSAC is pretty well-known out there."

As sequencing technology matures, GSAC organizers have worked to broaden its appeal, with entire days devoted to microbial genomics (see www.bio-itworld.com/news, DocFinder 6140) and genomic medicine (see www.bio-itworld.com/news, DocFinder 6157). Technological advances in genome sequencing closed out the meeting.

"I've chaired it for 16 years and I'll be chairing it next year." J. Craig Venter

While the "$1,000 genome" buzzword was conspicuously absent, sequencing by synthesis dominated the discussion among industry and academic researchers (for an excellent overview, see Shendure, J. et al. "Advanced Sequencing Technologies: Methods and Goals." Nature Reviews 5, 335-44; 2004).


A Need to FRET 
Stephen Quake, who recently moved from Caltech to Stanford University's bioengineering faculty, discussed fascinating research on single-molecule DNA sequencing by synthesis. On the same day, Quake was named one of nine recipients of the first NIH Director's Pioneer Award.

"DNA polymerase is a little Xerox machine," Quake said. "We set about building an assay to study this guy at the single-molecule level. The idea is very simple: You have a blank slide where you anchor your [single-molecule] primer template primer labeled with fluorescent dye. You add DNA polymerase, along with fluorescent labeled nucleotides, and then you'll end with DNA that has all these fluorescent tags on it. The goal is to try to image this process as it's happening."

Creating the right chemistry and microscope to distinguish such events is not so simple. Quake uses fluorescence resonance energy transfer (FRET), a process in which excitation is transferred from a donor dye molecule to an acceptor molecule.

Briefly, in the first round of synthesis, the incorporated nucleotide contains a "donor fluorophore," which will transmit energy to a nearby "acceptor" fluorophore if present. Subsequent synthesis rounds use acceptor fluorophores, which absorb the energy from the previously incorporated donor and turn on. This FRET signal is detected and imaged. It's not necessary to "see" juxtaposed nucleotides, only to know the time sequence of the images and compare them to one another.

"We've done the first single-molecule proof-of-concept sequencing experiments, and we've developed a set of assays that will be very useful for everything from use in new sequencing technologies to studying the basic properties of how polymerase operates," Quake said.

Many hurdles remain, but, Quake said, "If you extrapolate our densities to a 1-in-by-1-in area, that corresponds to 12 million different templates — sort of the ultimate shotgun method ... We think it should be possible to improve this by a factor of five or more." One startup, Helicos BioSciences, is working to commercialize the technology.




Tiny Bubbles 
Jingyue Ju, head of DNA sequencing and chemical biology at the Columbia University Genome Center, has developed a photo-cleavable nucleoside triphosphate that can be incorporated into a growing strand of DNA, imaged, and then cleaved by UV irradiation, making way for the next round of synthesis (Li, Z. et al. "A photocleavable fluorescent nucleotide for DNA sequencing and analysis." PNAS 100, 414-19; 2003). Ju suggested that sequencing by synthesis, if it became economical, would allow heterozygotes to be distinguished more effectively than traditional electrophoresis sequencing techniques would.

454 Life Sciences president and CEO Richard Begley brought discussion somewhat closer to reality. After two years of development, Begley was ready "to talk about a production system that runs every day." 454 will now adopt a two-pronged strategy: one aimed at whole-genome sequencing and another at comparative genomics for large groups of patients. The sequencing instrument will be available in early 2005.

454 has bet big on bead technology. After fragmenting DNA samples, a single strand is attached to a 3-micron bead. The beads are washed in oil with PCR reagents and become encapsulated in emulsion "bubbles," creating mini reactor vessels. Amplification produces 107 fragments per bead, Begley said.

GSAC Returns to Its Roots

To paraphrase Mark Twain (and J. Craig Venter), the reported death of the Genome Sequencing and Analysis Conference (GSAC) is premature, although a few changes are in the works. Read More

The beads are then placed in wells on a slide. Currently, there are three slide sizes: 300K wells, 860K wells, and 1.6M wells. Processing is highly automated, with imaging completed after each round of nucleotide incorporation. The current production run is four hours with 100-base reads. The instruments can do 24 million to 48 million bases per run.

"Our goal is to get it to the point where in a 24- to 48-hour time you can start from genomic DNA in the door to assembled genome out the door, no matter what the size of the organism. For viruses and bacteria, we do that today. For fungi, we hope to do it by Christmas. Sometime toward the end of 2005 or early 2006, we hope to be able to say the same for a human being," Begley said.

The computational power to capture images and run necessary informatics is substantial.

"We're working with Eugene Myers (UC-Berkeley and member of 454's scientific advisory board) to develop a massively parallel, short-fragment, super-efficient assembler, and it currently operates on a small cluster; it's being ported to the Xilinx field programmable gate array (FPGA) chip," Begley said. The chips can operate at 2 gigaFLOPS (2 billion floating-point operations per second) and use a "very dense" programming language purchased from a company in Europe.

"We have to simultaneously collect all the images. It all occurs on this chip during the wash cycle," Begley said. "During the very last wash, we are doing scaffolding on this chip against reference genome. It takes about 10 minutes for the scaffold bacteria against reference. The assembler, which we are just now getting to demonstrate for doing genome assembly, will be a callable subroutine that will eventually, we hope, work on one or two of these chips, in a single computer [housed in the base of the instrument]."

Reaching the "$1,000 genome" was the guiding principle of Agencourt Biosciences CSO Kevin McKernan's talk. Agencourt uses traditional capillary electrophoresis techniques but is developing bead-based systems, leveraging work by Harvard University's George Church.






PHOTO COURTESTY OF BOSTON UNIVERSITY FILE PHOTO