In a dramatic advance for alternative DNA sequencing technologies, scientists at 454 Life Sciences Corp. have essentially sequenced and assembled a complete bacterial genome sequence based on a mere 4-hour run on the company’s proprietary instrument.

454 Life Sciences' DNA sequencing system.

The analysis of some 25 million bases of DNA sequence using 454’s “sequencing by synthesis” nanotechnology approach allowed a team of more than 50 researchers to assemble almost the complete genome of Mycoplasma genitalium – some 580,000 nucleotides, or bases – with greater than 99 percent accuracy. The group, reporting in the online edition of Nature, claims a 100-fold increase in efficiency over conventional sequencing methods.

“This paper struck me as approaching one of the quantum leaps the National Human Genome Research Institute asked for in its vision for the future of genome research a few years ago,” says Nature senior editor Chris Gunter. “We all acknowledge that there needs to be cheaper, faster sequencing, and here's the first new technology since Sanger sequencing.”

“It’s completely analogous to personal computers displacing mainframes,” enthuses 454 founder and chairman, Jonathan Rothberg. “Now, anyone can have their own genome center. If you can miniaturize something, then everything gets cheaper and faster.”

Chemical Reaction
DNA sequencing chemistry has hardly changed since the establishment of the dideoxy sequencing method by Nobel laureate Fred Sanger and colleagues in Cambridge, England, back in 1977. Advances in automation from Applied Biosystems and other companies have permitted rapid improvements in sequence throughput and affordability, culminating in the completion of the Human Genome Project. But in recent years, companies such as Solexa, Helicos, and 454 have been developing new approaches that rely heavily on computer algorithms to virtually assemble hundreds of thousands of relatively short (about 100 bases) DNA fragments to produce the final sequence.

The genesis of 454 can be traced back to 1999, when Rothberg’s newborn son was rushed to hospital. The founder of CuraGen was left pondering what it would take to develop a new approach to DNA sequencing that could provide personalized genome information. He spun off 454 Life Sciences from CuraGen the following year.

454’s instruments – costing $500,000 apiece – are currently being tested at the Broad Institute, the J. Craig Venter Institute Joint Technology Center, the Wellcome Trust Sanger Institute, and other leading sequencing centers. The results in the Nature article explain why the new platform has aroused such intense interest.

The 454 approach involves shearing the starting material DNA using a nebulizer. Rothberg explains: “[We] nebulize the DNA into little fragments, shake it in oil and water, so each DNA fragment goes into a separate water droplet. So instead of bacteria, we separate the DNA into drops. Then we do PCR, so every drop has 10 million copies. Then we put in a bead, drive the DNA to the bead, so instead of the cloning and robots, one person can prepare any genome.”

The DNA-covered beads are loaded into the microscopic hexagonal wells of a fiber-optic slide, which contains about 1.6 million wells. In 454’s benchtop instrument, chemicals and reagents flow over the beads in the wells. Solutions containing each nucleotide are applied in a repetitive cycle, in the order T-C-A-G. Excess reagent is washed away using a nuclease, before a fresh solution is applied. This cycle is repeated dozens of times. 

As each base is incorporated into the DNA fragments on each bead, pyrophosphate is released, generating photons that are detected in quantitative fashion by a CCD (charge-coupled device) sensor on the base of the slide. Runs of a single base, e.g. A-A-A-A show up as four times the intensity of a single nucleotide position.

The 454 Protocol
Rothberg and colleagues detail in the Nature article the process for resequencing the genome of M. genitalium. After one person prepares the beaded DNA fragments on the fiber-optic slide (about 6 hours), the automated sequencing by synthesis reactions (42 T-C-A-G cycles) takes just four hours. With an average read length of 110 bases and 40-fold sequence coverage, and correcting for accuracy, the researchers covered 96.5 percent of the bacterial genome with 99.96-percent accuracy from a single instrument run.

“The referees felt that the improvement in sequencing technology was very important and would make big strides in the field, to the point of changing how sequencing centers are set up and run,” says Nature’s Gunter. “You know you have a good paper when it's in the review process and people unconnected to the paper approach you at meetings and say, ‘Oh, I heard you have that paper -- it's really exciting!’ And that's what happened with this one.”

The 454 authors concede that there are still key improvements to be made in read length and accuracy. While the average read is 100 bases, the instrument has been able to produce accurate runs of 200 bases, and occasionally even 400 bases. The accuracy with genomic DNA samples is lower than for test fragments, but that is attributed to problems with the templates, not the sequencing technology per se. And the accuracy of individual sequence reads relies on replication and redundancy to match that of more traditional Sanger methods.

Noting parallels with Moore’s Law, the authors conclude: “Future increases in throughput, and a concomitant reduction in cost per base, may come from the continued miniaturization of the fibre-optic reactors, allowing more sequence to be produced per unit area – a scaling characteristic similar to that which enabled the prediction of significant improvements in the integrated circuit at the start of its development cycle.”

There are 56 co-authors on the Nature article, all but three from 454, based in Branford, Conn. Among the other contributors is Gene Myers, the Berkeley geneticist who headed the algorithm assembly team at Celera Genomics.

Margulies, M. et al. “Genome sequencing in microfabricated high-density picolitre reactors.” Nature. Doi:10.1038/nature03959