Little and Large of Sequencing

New machines from Ion Torrent, PacBio headline AGBT.

March 16, 2010 | MARCO ISLAND, FL—New instrumentation from Pacific Biosciences and Ion Torrent Systems, along with a preview of a forthcoming “3rd-generation” single-molecule system from Life Technologies, dominated a superb conference at AGBT.*

Looking to repeat the magic of 454 Life Sciences, which debuted five years ago, Jonathan Rothberg, founder of Ion Torrent Systems, presented a $50,000 machine that does semiconductor sequencing—dispensing with lasers, optics, and fluorescence. To drum home the point of its portability, two Ion Torrent staff carried in the prototype “Personal Genome Machine,” which had been sequencing DNA in a nearby hotel suite, during Rothberg’s presentation.

Rothberg insisted to Bio•IT World this was not “next-generation sequencing,” which was only empowering for a few. Likening next-gen technology to the minicomputer, Rothberg said his ultimate goal was to develop the sequencer equivalent of the personal computer. He credited Broad Institute sequencing director Chad Nussbaum with coining the term: “post-light sequencing.”

At the heart of the Ion Torrent instrument is a semiconductor chip, initially containing 1.4 million sub-microscopic wells. Details of the sample prep were not disclosed. Because no imaging is required and the location of the wells is fixed and known, the demands on data storage and management are greatly reduced. Instead of a mini-supercomputer or blade center, the accompanying IT is a little more than a laptop.

Acid Music

The system works by cycling in nucleotides and directly measuring the pH change caused by the release of a hydrogen ion during the reaction. The pH change puts a charge on a sensing layer directly under the wells, which is turned into a voltage. If two identical bases are incorporated, then the chip detects a doubling of the pH change. Rothberg says it is important to accurately call eight consecutive polymeric bases, but beyond that, any repeat lengths are probably not biologically relevant.

Unlike other systems that feature modified bases and engineered or modified enzymes, Ion Torrent uses natural unmodified bases and polymerase. It’s the simplest sequencing in the world,” says Rothberg. “One well, one sensor, one read.” Currently, a run takes about an hour, during which time Rothberg claims it can read about 100-200 bases, with good quality sequence available from about half the wells on the chip.

“We’re not competing with [the Illumina] HiSeq,” said Rothberg. “This chip is not for whole genome sequencing of humans.” Rather, he sees a host of applications in research and clinical settings—bacterial genomes, gene expression, target exons, structural variations in cancer, metagenomics, etc. Rothberg presented preliminary data on a viral and bacterial genome, assembled with the aid of DNAStar.

The Personal Genome Machine will officially launch later this year. Rothberg announced he will give away two machines for the best sequencing application ideas in April.

For now, the read lengths are about 100-200 bases in 1-2 hours, but Rothberg says, “there’s no reason it couldn’t go to 1000 bases.” As per Moore’s Law, increasing the number of reads on the chip appears a formality. One million reads would cost the same as 7 million, he said, adding that as the reads get smaller, the sequence gets faster and more accurate.

Splash Down

Pacific Biosciences introduced what it called the first “third-generation” DNA sequencing instrument. “It’s really the world’s most powerful real time, single-molecule microscope,” said PacBio CEO and chairman Hugh Martin, who hailed the machine as “a quantum leap” for the field. “We sold the ten beta units just like that—fully paid for!” Those instruments will ship to North American customers by June.

The $695,000 PacBio RS weighs about 1900 pounds and is 6 1/2 feet wide and 29 inches deep. The instrument is accompanied by a blade center, which sits next to the main instrument. Much of the instrument’s girth is taken up by the robotic sample mixing and staging system. The base is packed with four high-speed cameras, optics, and a carbon-fiber stage good to +/- 5 nm in six dimensions. Martin admits: “You need a pretty heavy duty floor.”

Martin defined 3rd-generation sequencing as “everything that 2nd gen is—throughput, cost per base, etc.—with the addition of very long read lengths, extremely low reagent or consumable cost and very fast run times.”

The PacBio RS is what one might expect from a company that has attracted $260 million and employs more than 300 staff. The sample prep takes place in about six hours, in which the DNA is sheared and converted with SMRT adapters into a topologically closed circle for consensus sequencing. The DNA template is complexed with the polymerase before loading onto the machine.

A separate drawer houses up to 96 SMRT (single molecule real time) cells. Each SMRT cell holds 80,000 zero-mode waveguides (ZMWs). The SMRT cells are lined into strips of eight (dubbed ‘8-packs’); 12 of these strips can be loaded at a time. Each SMRT cell is individually sealed, so an instrument run could involve just a single unit. The run time for a SMRT cell is about 15 minutes, with the polymerases running at about 1-3 bases/second. “As soon as you start collecting data, you start processing. You can make iterations or changes in real time,” says PacBio’s Geoff Otto.

A strobe method can be used for longer read lengths of 3-10 kilobases, alternating short reads with ‘dark’ segments to minimize photo-damage to the polymerase. Martin’s goal is to create “a sunburn-proof enzyme” that in 2-3 years, will produce read lengths of 40,000 bases and more than 100,000 bases under strobe conditions.

The blade center handles the robotics and real-time data processing. Edwin Hauw, senior project manager, said PacBio’s software package “covers everything in the sequencing workflow from run design to instrument loading, run monitoring, primary and secondary analysis.”

The blade center handles real-time data collection and signal processing using a hardware accelerator. There are four blades; each blade has dual Intel Nehalem quad-core processors and 12 Terabytes, with a total of 192 Gigs RAM. One blade handles the robot, the other three data analysis, movie-to-trace, and trace-to-pulse-to-base call. There is sufficient storage for 24 hours worth of base calls and quality values.

At full release later this year, he said the average read length will be 1000-1250 bases, with 5% reads between 3-5 kb, around 1,000 reads per cell. “We’re the first mover. There’s no one else out there in 3rd gen.” Martin controversially elected against shipping to early access customers outside North America, but has accelerated plans to ship abroad later this year. Even with future upgrades, the current machine will not be the one that delivers the ‘15-minute genome,’ as PacBio founder Stephen Turner claimed two years ago. A V2 instrument will reach that target in 2014.

Exciting preliminary applications on the PacBio platform were presented by collaborators at Stanford and The Genome Center, Washington University.

Lights, Camera, Action

While Life Technologies continues to push upgrades to its SOLiD system, it unveiled early results on a new single-molecule platform using Quantum Dots. Joe Beechem presented trace data showing fluorescence energy transfer from a Q-dot, attached to a DNA polymerase, to a fluorescently tagged nucleotide in the active site. Life Technologies hopes to be working with early access partners later this year.

There was much cool science too. Vanessa Hayes presented work on southern African genomes including Archbishop Desmond Tutu, interestingly sequenced and validated on all three leading platforms. Complete Genomics announced strong progress with 50 customers and 500 orders for its whole-genome sequencing service. Patrice Milos showed how Helicos is fine-tuning its system for direct RNA sequencing.

*Advances in Genome Biology and Technology; Marco Island, Florida. February 25-28, 2010.

This article also appeared in the March-April 2010 issue of Bio-IT World Magazine.
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