NEW YORK (GenomeWeb News) – In a paper appearing online today in Science, researchers from Pacific Biosciences published a proof-of-concept study using Pac Bio’s single molecule, real-time DNA sequencing technology.
Pacific Biosciences, a privately held company based in Menlo Park, Calif., has been chasing real-time single molecule sequencing since 2004. It has developed a system for hijacking single DNA polymerase molecules, immobilizing them beneath a detector, and using them to incorporate labeled nucleotides. The fluorescent tags are removed from these nucleotides as they are incorporated into a growing DNA strand, producing unmodified DNA and giving off signals detected by a sensor.
With the new publication, Pac Bio illustrates the feasibility of sequencing with the system, which detects the enzymatic incorporation of four kinds of fluorescently labeled deoxyribonucleoside triphosphates, or dNTPs, using a device called a zero-mode waveguide, or ZMW, nanostructure array.
By introducing site-specific modifications in a DNA polymerase called Φ29, the researchers developed an enzyme that can efficiently use labeled nucleotides. Fluorescent tags are linked to the terminal phosphate of dNTPs so that they are released during bond formation.
“DNA sequence is determined by detecting fluorescence from binding of correctly base-paired (cognate) phospholinked dNTPs in the active site of the polymerase,” the authors explained. “A fluorescence pulse is produced by the polymerase retaining the cognate nucleotide with its color-coded fluorophore in the detection region of the ZMW.”
That approach lets researchers directly observe DNA polymerization as it happens, with thousands of single-molecule sequencing reactions measured in parallel. And, the researchers said, along with sequence data, the technique may also let scientists capture other types of information besides DNA sequence.
“In addition to the sequence, the real-time aspect of our approach generates unprecedented information about DNA polymerase kinetics that will allow other uses of the technology,” Pac Bio Founder and Chief Technology Officer Stephen Turner, the paper’s senior author, and his colleagues wrote. “Because the system reports the kinetics of every base incorporation ... the system can be used today to investigate kinetics of DNA polymerization with unprecedented resolution and speed.”
The researchers tested the system in a series of two-color and four-color DNA sequencing experiments. Overall, the team found that they could generate consensus sequences at 15-fold coverage with a median accuracy of 99.3 percent.
The researchers found no discernible sequence context bias and “no systematic error beyond fluorophore-dependent error rates.” They reported that deletions were the most common type of error owing to base incorporations that were too short to detect reliably.
“The present level of accuracy can produce alignment and consensus adequate for re-sequencing applications,” the authors wrote. “However, it would create challenges for de novo assembly or alignment into highly repetitive DNA.”
The authors said that it should be possible to improve the accuracy of the system — for instance, by improving enzyme kinetics, increasing fluorophore brightness, or using circular consensus sequencing. They also noted that sequencing should become more multiplexed as camera technologies develop and are able to monitor more ZMWs.
“As these technologies evolve, it will be possible to provide later generations of this instrument with multiplex commensurate with current step-wise sequencing systems,” the authors concluded. “Combining this level of multiplex with the high intrinsic speed and read length of single-molecule real-time DNA sequencing will enable low-cost rapid genome sequencing.”
This September, Pac Bio CEO Hugh Martin said that the company plans to kick of its early-access program in the first half of 2009. In the second half of next year, Pac Bio hopes to have customers for its in-house production machines. The company has said that it is aiming to bring its platform to the market by the second half of 2010.
Earlier today, the company announced that it had raised $20 million in funding from new investor Blackstone Cleantech Venture Partners, bringing its total financing since it was founded to $193 million.