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PacBio, Northeastern Using NHGRI Grant to Improve SMRT Sequencing by Tweaking DNA Entry into ZMWs

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Researchers from Northeastern University and Pacific Biosciences plan to use an $825,000, three-year grant from the National Human Genome Research Institute to reduce the DNA input requirements and cost of PacBio's sequencing technology.

The team - led by Pacific Biosciences' Jonas Korlach and Northeastern physicist and chemical biology professor Meni Wanunu - will use approaches developed in Wanunu's lab to increase the efficiency and decrease the cost of PacBio's Single Molecule Real Time sequencing by manipulating the rate and exclusivity with which molecules enter each of its zero-mode waveguides.

The PacBio/Northeastern group is one of six projects awarded a total of $19 million in grants last week under NHGRI's Advanced DNA Sequencing Technology Program.

Pacific Biosciences' founder and chief technology officer Steve Turner told In Sequence that the company isn't sure exactly how much the project will be able to reduce the cost of its sequencing, but that the team expects to be able to increase efficiency at least three-fold through one arm of the effort.

"Part of the function of these grant-funded activities is to enable commercial organizations like us to go after things that are bigger gains and also riskier, so it's hard to predict how big the gains could be, but they could be very large," he said.

Turner said the project will be based on methods Wanunu developed using electric fields to control DNA molecules. A main goal will be to use Wanunu's approach - which he described in a paper in Nature Nanotechnology in 2010 - to control the distribution of molecules into PacBIo's waveguides, making it so that after one molecule has "entered the arena," Turner said, "its presence would close the door to any others entering."

"Today we have to use a statistical distribution and rely on the randomness of binding to ensure that sometimes we see only one polymerase sequencing [in each waveguide.] The rest of the time we either get none or more than one, and those are not productive," Turner said. "So the idea that you could have a molecule that could close the door behind it means we can get essentially all the functional sequencing units to be providing data."

Wanunu told In Sequence that his method for closing the proverbial door after one molecule has entered a waveguide will involve putting a nanopore at the bottom of the waveguide wells.

"If you think of the waveguide as a well, we are going to put a single binding site in each well for a polymerase, and the way we are going to do that is by having a pore [50 times smaller] there," Wanunu said. "This would increase the functionality from about 37 percent to theoretically 100 percent."

Turner agreed that based on the best performance circumstances the company achieves currently, this nanopore door-closing could improve the throughput, and therefore the cost of sequencing, by a factor of three.

But insuring that each waveguide only contains a single molecule is only one way the group hopes to use Wanunu's electrical manipulation approach to increase efficiency and reduce cost.

Turner said the electrostatic control method will also allow for much lower concentrations of input DNA. "One of the things that happens when you use electric forces to move a molecule [is that] you can direct it from much farther away, [meaning] at a much lower concentration," he said. "There we are not just talking about [reducing input] by a factor of three; it could be a factor of several hundred or 1,000."

"Think of the waveguide as something you are leasing," Wanunu said. "Each has a certain lifetime, and in that time we want to sequence as many strands as possible. You can only go so far if your DNA doesn't enter the well - so that's the way we're going to reduce the cost."

According to Turner, these "quantitative" gains could take some applications for the PacBio that are right at the border of being viable and "push them over the edge."

For example, he said, "it would take a cancer biopsy sequencing experiment where there wasn't enough DNA and make it so there was enough DNA ... or take a system where economically you wouldn't have enough coverage ... and by cutting cost to a third it would make it economically feasible."

Forensics and archaeology applications could also be better enabled by lower DNA input requirements, he added, as well as sequencing highly repetitive regions of DNA where researchers may require longer read lengths than can be amplified with PCR.

Turner said the company sees potentially even more possibilities in Wanunu's electrostatic focusing approach for controlling the placement of individual molecules. "Being able to nominate a particular molecule from a group to go over someplace to get sequenced ... that's a whole other paradigm [than] what we are used to," he said.

"Right now we treat everything like the contents of a lawnmower bag. Someday we'll be able to be more like a gardener."

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