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PacBio Shows Proof of Principle for Methylation Sequencing, Direct RNA Sequencing

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By Julia Karow

This article was originally published Sept. 17.

COLD SPRING HARBOR, NY – Pacific Biosciences has demonstrated that it can use its single-molecule real-time sequencing technology to identify methylated DNA bases, and to directly sequence RNA molecules. It has also applied strobe sequencing, a sequencing mode that generates multiple gapped reads from a single DNA strand, to map complex structural variations in human fosmid DNA.

At the Personal Genomes conference at Cold Spring Harbor Laboratory last week, PacBio Chief Technology Officer Steve Turner showed that the company's platform, which measures the incorporation of fluorescently labeled nucleotides into DNA by a polymerase in real time, can distinguish methylated from unmethylated bases for two types of nucleotides.

In principle, this will allow users of the platform to simultaneously determine the DNA sequence and its methylation status. By comparison, current sequencing platforms require DNA to be specially prepared to interrogate methylation sites, for example using bisulfite or methylation-specific antibodies.

The detection hinges on differences in the kinetics of the enzymatic reaction, depending on whether a methylated or an unmethylated nucleotide is incorporated, he explained. The ability to distinguish between the two improves if the same molecule is sequenced several times.

So far, the company has shown that it can distinguish naturally methylated adenosine in E. coli DNA from unmethylated adenosine in DNA from the bacterium that has been amplified in a test tube. Methylated adenosine, he said, appears to interfere with base-pairing during the sequencing reaction.

Detecting changes in the kinetics associated with methylated cytosine is “a little harder,” he said, but the company just recently succeeded in distinguishing methylated from unmethylated cytosine, although the signal was not as good as for adenosine. Turner did not mention whether methylation sequencing will be available at the time of the commercial launch of PacBio’s platform, planned for the second half of next year.

In addition to methylation sequencing, the company has also shown proof-of-principle for directly sequencing RNA. This application requires an RNA-dependent type of polymerase, such as reverse transcriptase. The rate of unproductive binding events of nucleotides is higher than for DNA sequencing, Turner noted. So far, the company has been able to read synthetic RNA templates that consist of alternating adenosines and uridines.

Turner said that direct RNA sequencing will not be available at launch, but is expected to be added within a year after the platform is released.

He also mentioned how the company has started to apply strobe sequencing, a mode of sequencing where instead of a single read, the platform generates several shorter reads from the same molecule that are interrupted by “dark” stretches of DNA of a defined size (see In Sequence 5/12/2009).

At present, the technology produces a distribution of reads with an average read length of 1,000 base pairs. However, each of these reads can be split up into several shorter “strobed” reads that cover a footprint of several kilobases of DNA.

Read length is currently limited by photochemical effects that inactivate the polymerase, but Turner said the company is “hard at work” to eliminate these effects. At that point, read length would only be limited by the enzyme itself and could potentially increase to dozens of kilobases, he said.

In a collaboration with Evan Eichler at the University of Washington, an expert in structural variation, PacBio has analyzed human fosmids using strobe sequencing. This allowed the researchers to analyze insertions spanning up to several kilobases in size and to more accurately resolve breakpoints, which would not be possible using conventional paired-end reads and a library with a single insert size. “It’s a powerful way to solve complex tangles,” Turner said.

The company currently has 12 prototype instruments for in-house research as well as collaborative work. These use chips with about 3,000 wells, or zero-mode waveguides, about a third of which are occupied with a single polymerase each.

The commercial instrument will have significantly more ZMWs, but the company is not yet disclosing that number. Chips for the instrument will be “the price of a nice dinner,” Turner said.

Over the next three to four years, the company expects sensors will become available that will enable it to run a million ZMWs per chip.

Turner said customers can expect a “rapid expansion of capabilities over the lifetime” of the first instrument generation. The second generation, he predicted, will be capable of sequencing a human genome at high coverage on a single chip.

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