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By Kevin Davies

March 1, 2008 | MARCO ISLAND, FL – On the second evening of the ninth AGBT conference*, a little known company, Pacific Biosciences, sponsored a beachside fireworks display to raise awareness among 600 delegates as it emerged from stealth mode.

It needn’t have bothered.

PacBio founder and CTO Stephen Turner’s closing presentation was unquestionably the talk of the meeting. Together with the launch of a low-cost, open source sequencing instrument called the Polonator, and Illumina’s announcement of a complete $100,000 human genome assembly, the ninth AGBT conference had more than its share of highlights.

15-Minute Genome
“How cool was that?!” purred Washington University’s Elaine Mardis, following Turner’s talk. Evolutionary geneticist Jonathan Eisen (on his Tree of Life blog) said Turner’s “technology is the first I have seen that has shown some results and that could really lead to the $1000 human genome.” And Yu-Hui Rogers, scientific director of the J. Craig Venter Joint Technology Center, hailed the technology as “potentially revolutionary.” Such sentiments were endorsed by many other academics, not to mention scientists from most of the current crop of next-generation companies, possibly bracing for extra competition a few years down the road.

Turner predicted that within five years, PacBio’s technology will be able to produce a raw human sequence in less than three minutes, and a complete, high-quality genome in just 15 minutes. Genome scientists have heard such claims before, but Turner’s preliminary data left most of the audience believing that this approach could be truly disruptive.

The company’s chairman and CEO, Hugh Martin, a former telecommunications executive, told Bio•IT World that a commercial instrument won’t be ready until 2010 or 2011. PacBio has raised almost $80 million thus far, and is looking to raise a lot more to finance that work. Nevertheless, Martin told The New York Times, “When we’re ready, we’re just going to win the [genomics] X Prize.”

Seeing the Light
PacBio was founded in 2004 — the company was originally called Nanofluidics — but the technology dates back to Turner’s days as a grad student and postdoc at Cornell University. The SMRT (single molecule, real time) system monitors the procession of a DNA template as it engages with a single DNA polymerase enzyme. Using four fluorescently tagged nucleotides, the system images each nucleotide as it is snagged by the enzyme prior to incorporation in the growing strand. The polymerase is tethered to the bottom of a zero-mode waveguide (ZMW) — a sub-microscopic, 20-zeptoliter well that the company claims is “the world’s smallest detection volume.” All this happens at a speed of about 10 bases/second (in nature, the polymerase moves 50-80 times faster).

Using the ZMW concept that Turner and his former Cornell colleagues, physicists Harold Craighead and Watt Webb, published in Science in January 2003, the PacBio method ingeniously illuminates the area around the tethered enzyme, while leaving the unincorporated fluorescent bases floating in the dark. The light generated by each nucleotide as it is snagged by the polymerase is recorded by a CCD camera, using a prism to separate the colors and thus identify each nucleotide.

Turner likens the principle to the small holes in the mesh screen door of a microwave oven, which do not allow the longer microwave radiation to pass through. As he told Bio•IT World: “If you shrink both the radiation wavelength and the holes down to the nanoscale, so the wavelengths go to visible light of 500 nm, and the holes are just a few tens of nanometers in diameter, the result is that if you illuminate the hole through the transparent substrate that’s holding it, light that impinges upon the circular aperture of the hole can’t pass through the hole. So all of these nucleotides, while close at hand and accessible to the polymerase, are in the dark and don’t contribute to the background noise.”

Meanwhile, the enzyme sits at the base of what Turner descriptively calls “the evanescent decay zone of the device,” allowing his team to specifically detect the emanating fluorescence from the enzyme-bound nucleotide.

Turner presented preliminary data on synthetic DNA templates. He presented CCD images of a grid of 1000 ZMWs on a chip smaller than a pinkie fingernail, which burst into fluorescent life when all the necessary ingredients are presented to the enzymes sitting in each well. That’s a throughput of 36 megabases/hour. (The video had to be slowed down, because the human eye wouldn’t be able to register the images in real time.)

“No-one’s ever seen 1000 polymerases making DNA before in real time,” says Martin proudly.

Advantages of the SMRT system include the location of the fluorescent tags (at the end of each nucleotide), such that they are removed before the base is incorporated into the growing DNA strand. The read lengths should be comparable to Sanger sequencing, thus avoiding the bioinformatics challenges of assembling very short reads. And the system will have no moving parts, aside from the polymerase itself, once a run is started.

Although the SMRT system is far from perfect, Turner presented readable sequence traces from known DNA templates, as well as the ability to derive consensus sequences by using circular templates that are read multiple times by the same enzyme. Turner says PacBio is launching its first genome sequencing project, after which it will have a much better sense of the accuracy of the system. That will be improved by ongoing studies on the directed evolution of the polymerase enzyme.

PacBio is still years from debuting its instrument, but Turner outlined plans for future enhancements that include (1) producing a chip with 1 million ZMWs; (2) increasing the speed of DNA polymerization from 10 to 50 bases/second; and (3) using a 20-megapixel CCD camera with on-chip magnification to make it single photon sensitive. Those enhancements are expected to deliver a throughput in five years’ time of 100 Gigabases/hour.

“This is what is required to get genome sequencing into routine medical practice,” says Turner. “It is disruptively faster than current... technologies. Instead of being hours per base, it’s bases per second.”

The Rise of the Polonator
There was no data from the other new platform unveiled at AGBT, but considerable interest in seeing the Polonator up close. This instrument is based on protocols developed by George Church’s group at Harvard Medical School and manufactured by Danaher Motion.

Kevin McCarthy, CTO for Danaher Motion, enthusiastically intoned visitors to “Lay your hand upon the sequencer.” Sure enough, waving in front of a motion sensor cues the Polonator to hum into life.

The instrument is based on the polony and ligation chemistry methods developed by Church and colleagues. In April 2007, McCarthy arranged to give a presentation to members of Church’s lab. He recalls: “My driver got lost on the way down, we had a hard cut off at 12 noon. We got there late, and basically I had 15 minutes. Stand and deliver. Apparently I did! It’s been all uphill since then.”

McCarthy had argued that his firm could dramatically improve the Church lab’s existing design plans and engineering. “For example, there was a $20,000 autosampler next to the instrument. ‘What’s that doing?’” McCarthy asked. “I think I can subsume that function and bring it inside the instrument in a much better way.” He didn’t meet Church himself until last August. But six months later, the first Polonator was being shipped to the Church lab, with the Broad and Max Plank Institutes to follow.

The Polonator sells for $150,000 — a snip compared to other next-gen platforms — although McCarthy concedes margins are small. It contains many examples of Danaher engineering, such as motors and a Leica lens and objective, along with a specialized $25,000 electron-modifying CCD camera from Hamamatsu that McCarthy says “is worth every penny.”

McCarthy says a high “level of obsession permeates this entire piece of hardware.” He adds: “I can strip this machine down to raw hardware in about 15 minutes… It’s eminently serviceable, and more to the point, eminently upgradeable.”

Aside from cost, the other appealing aspect of the Polonator is Church’s open-source approach. “We’re drinking the cool aid! George is a visionary,” McCarthy says. “We’re enabling the vision. His vision is, ‘I want this adopted as quickly as possible. I need an engine for the Personal Genome Project.’”

The Church lab will be making its pair of Polonator software source codes freely available for download, allowing users to improve and extend as they wish. One program runs image acquisition and controls the instrument, while the other is responsible for image processing and real-time base calls. These functions are handled by separate dual-core CPU computers.

The open-source philosophy extends to the wet side as well. Originally, Danaher was not going to supply reagents. “People were like, ‘Look, I don’t want to deal with all the [supplies],’” says McCarthy. So Danaher agreed to produce kits, but is encouraging customers to seek better deals elsewhere. He says the Polonator currently uses a novel, low-cost PCR polymerase, a very low-cost ligase, “and all the fluors are license free — freedom fluors!”

Each of two flow cells contains 18 wells, which hold about 60 million DNA-coated beads, for a total of some two billion beads. “As Carl Sagan says, that’s a large number,” jokes McCarthy. “One is undergoing biochemistry, while the other is being imaged.” A completely automated run takes about 80 hours, producing 10 gigabases of sequence per run. Each read consists of 28 bases, 14 from each paired tag in every DNA fragment.  “That’s just what we’re going out of the gate with,” says McCarthy, but he expects significant improvements in the near future.

Another Week, Another Genome
With so much made of the two new platforms, the presentation by Illumina’s David Bentley of the complete assembly of the world’s first African genome was somewhat overlooked.

Bentley, based at the former Solexa headquarters near Cambridge, U.K., said that in a span of 6.5 weeks at the end of 2007, Illumina successfully sequenced and assembled the genome sequence of one of the original HapMap donors, for about $100,000. The work was done primarily with paired reads of 35 bases in length, although Bentley said that 50-base reads are becoming more standard. Recent improvements in the Genome Analyzer, including larger channels and improved optics, allowing for shorter run times, enabled the work to proceed, following a pilot project on the human X chromosome.

To sequence the African male DNA sample, Illumina used two libraries containing 200-base pair (bp) and 2-kilobasepair inserts. Performing 27 runs on the Genome Analyzer, Bentley’s team produced almost 77 Gigabases of DNA sequence, an average of 3.27 Gb per run, spanning more than 90 percent of the genome.

Bentley said that the sequencing revealed more than 3.7 million single nucleotide polymorphisms (SNPs), including more than one million novel polymorphisms. The work also identified a number of structural variants, large deletions, and chromosomal rearrangements. Public data release is expected very soon.

 

In the News:

454 published a paper in the New England Journal of Medicine identifying an arenavirus as the culprit underlying the sudden, mysterious deaths of three organ transplant patients in Australia;

Applied Biosystems announced Geospiza and GenomeQuest had joined its SOLiD software development community;

Helicos sold its first HeliScope, priced at $1.35 million, to an undisclosed customer.

 

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 This article appeared in Bio-IT World Magazine.
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