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Intel, PacBio Researchers Among Team Taking on Exploratory Project Pairing SMRT Sequencing with Electronic Tags

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A research team based at Intel Corporation, Pacific Biosciences, the University of Twente, and Columbia University is embarking on a project examining the feasibility of doing single-molecule, real-time sequencing with electrochemical tags.

Last month, the effort secured just shy of $5 million in funding through the National Human Genome Research Institute's Advanced DNA Sequencing Technology Award Program to support the project over four years (GWDN 9/14/2012).

"It's a scintillating idea to combine some technologies that Intel has in [its] portfolio with technologies that we have in ours," PacBio founder and Chief Technology Officer Stephen Turner told In Sequence.

In particular, the group is looking at whether they can come up with distinct electrochemical tags for each of the four DNA bases so that electrical rather than fluorescent signatures are released by each base during the SMRT sequencing process

"Within the [research] program, we would be using distinguishable electrochemical tags as a means of identifying what nucleotides had been or [were] being incorporated," Turner explained. Those electrochemical tags would then be "the substitute for the spectroscopic means that we use today with the optics that read detectable labels."

If such a strategy pans out, it might be possible to produce highly parallel SMRT systems that function without fluorescence and the corresponding optical hardware. This would help increase the scalability of the system, as well as simplify it and subsequently decrease costs.

Other sequencing companies are also looking beyond fluorescence-based sequencing. Life Technologies' Ion Torrent systems use a semiconductor sequencing-by-synthesis method, based on the detection of hydrogen ions released during DNA polymerization. The Ion Torrent systems, though, are not single molecule. Illumina has also said that it is looking to develop sequencing chemistries that do not rely on optical detection, but has not provided a timeline for commercial launch of such chemistries (IS 2/14/2012).

Still, Turner emphasized that the PacBio project is at a preliminary research stage and that PacBio's commercial efforts are still very much focused on optimizing and integrating its fluorescence-based SMRT technology.

"At this time the Intel collaboration is really an exploratory research program," he said, noting that the relationship between the firms stretches back some time.

For instance, Intel's venture capital arm, Intel Capital, was an institutional investor in PacBio during some of the sequencing firm's funding rounds (IS 8/18/2009).

"This opportunity presented itself to take some technologies that they've been noodling on and some technologies that we've been noodling on to see how they play together," Turner said. "And that's really what this grant application is," he said. "[I]t is, in the end, a combination of two existing technologies."

Also participating in the NHGRI-funded effort are researchers from Columbia University and the University of Twente in the Netherlands, who bring experience in circuit design and expertise related to single-molecule physics to the mix, respectively.

"We're very excited to get started and glad to be working with [the National Institutes of Health] and the awesome four-way team," project co-leader Madoo Varma, head of Intel's integrated biosystems lab, told IS.

Intel has an ongoing internal research program related to DNA sequencing, she noted. Earlier this year, for example, Varma and her colleagues reported on an electrical, label-free method for detecting DNA polymerase reactions with modified field-effect transistor devices (IS 2/28/2012).

And the Intel team has been developing chemistry related to doing sequencing-by-synthesis approaches with electronic tags.

In particular, Varma explained, the use of electronics offers the potential for enhanced scalability in a single-molecule sequencing setting — an area that can be challenging for optical sequencing systems.

"Ideally, single-molecule SBS can be massively-parallel and real-time, operating at synthesis rates as high as 1 [millisecond] for DNA polymerase," Varma, Turner, and colleagues wrote in their NHGRI sequencing award abstract. "[H]owever, complex optics required to collect photons efficiently make scaling of the platforms to high densities difficult."

"A promising route for overcoming the challenges to optical techniques is bio-electronic detection," they added.

Specifically, the team proposes using electrochemical rather than fluorescent tags in the SMRT sequencing setup, so that as each nucleotide produces a characteristic electrical signature it gets incorporated into the growing DNA strand by the polymerase enzyme.

In addition, researchers propose using a process known as redox cycling to amplify the signal produced by each reduction-oxidation, or redox, active tag, which would theoretically be detected by nanogap transducers integrated using a complementary metal-oxide semiconductor, or CMOS, circuit approach.

"It's basically a label detection technique that is conceptually analogous to fluorescence detection, but uses an electron transfer with a nanogap electrode rather than a photon emission and detection as a means of detecting that there was something there," Turner explained.

The researchers did not disclose further details of the study, such as the identity of the electronic tags being considered or tested. But Varma noted that the Intel team has already been working with one electronic tag and is in the process of developing others.

Researchers suspect that combining SMRT sequencing with the scalability of CMOS integrated electronics could potentially allow for more pronounced parallelization and multiplexing. But there may be other theoretical benefits as well, Turner said.

For instance, being able to detect electrical rather than fluorescent signals would also obviate the need for the illumination optics used in an optical system, which would streamline the system and bring down costs.

In principle, an electronic SMRT system should be suitable for sequencing not only DNA, but also RNA. Since the project is still at an extremely preliminary stage, though, members of the team cautioned that proof-of-concept experiments are needed to explore the feasibility and capabilities of such a system.

For instance, it is unclear what sorts of speed, throughput, or accuracy might be possible with an electronic SMRT system. Similarly, members of the team were hesitant to make estimates about the read lengths that may be attained with such a system.

For his part, Turner argued that by the time it comes to fruition, the read lengths produced by electronic SMRT sequencing might not be all that different than those that can be attained with future iterations of the company's current sequencing strategy.

That's because advances are continuing to be made to curb polymerase enzyme photo damage in the fluorescence-based system, he noted.

PacBio also announced plans last week to launch new read length-extending chemistry for its sequencing system in the fourth quarter of this year (see story, this issue).

"By the time this technology would be ready, I think that it would be at parity read length-wise and that that would mean maybe it could be in the 50,000 base region, maybe even higher," Turner said.

"One doesn't know," he added, noting that "these enzymes, in nature, are capable of going significantly further than that."

As such, he speculated that an electronic tagging system, if successful, might not necessarily provide performance enhancement on the read length front, but rather an overall simplification of the system and a means for multiplexing.

"Any time you can remove a component from a system, the resulting simplification reduces costs and there are advantages there," Turner said.

In the nearer term, though, PacBio is focusing its commercial efforts on improving its fluorescence-based system.

This summer, the company announced that it was collaborating with the Belgian non-profit nanoelectronics firm Imec to develop microchips that would help in scaling up and multiplexing the SMRT system (IS 7/24/2012).

Turner emphasized that that partnership is focused on "integrating, miniaturizing and reducing cost and size, and increasing the performance and speed of the existing technology," adding that the group is "very pleased" with the progress being made on that front so far.

"The Imec deal is our principle effort towards a compact, monolithically integrated device that would have all the functionality that we're looking for," he said. "That [project] is based much more closely on the technology that we're using — the fluorescent-labeled technology."

On the other hand, because the electronic tagging project is "at the very beginning," Turner explained, "it's too soon to be making accommodations in our principle product development for that."

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