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PacBio, Northeastern Team Combine Nanopores, ZMWs for Low-Input, Long-Molecule Sequencing


SAN FRANCISCO (GenomeWeb) – Building off previous work, researchers from Pacific Biosciences and Northeastern University have demonstrated that combining nanopores with PacBio's sequencing system can help preferentially load long DNA molecules and enable lower amounts of input DNA.

The study, published today in Nature Nanotechnology, is one result from research supported by a three-year $825,000 grant from the National Human Genome Research Institute. In 2014, the group published the first results of that work, demonstrating that the hybrid nanopore/zero-mode waveguide (ZMW) configuration could be built and that DNA could be loaded onto the sequencing system through that configuration.

Now, the team has demonstrated that the devices are active, that DNA can be sequenced and read out, and importantly, that less input DNA is needed than typically required for PacBio sequencing.

"It's still a proof of concept," said Meni Wanunu, an associate professor of physics at Northeastern and senior author of the study. The next step is to demonstrate scale — constructing wafers that include a pore for every ZMW, as opposed to making each pore separately, he said. In addition, he said that his team plans to focus on developing direct RNA sequencing applications.

Jonas Korlach, PacBio's chief scientific officer, declined to discuss whether the firm plans to incorporate nanopore ZMWs (NZMWs) into its commercial platform. He said that the Nature Nanotechnology study "does not in any way reflect any projections about commercial implementations or features of the current or future SMRT sequencing platforms."

Wanunu said that the goal of the collaboration with PacBio was to figure out a way to decrease the amount of input DNA needed for the system and to preferentially load longer molecules of DNA.

One challenge with SMRT sequencing, he said, is loading longer DNA molecules into the ZMW, a 100-nanometer sized well. Currently, DNA templates must bind to the ZMW through biotin-streptavidin chemistry, and the molecules make their way to the ZMW through diffusion, a process that favors shorter fragments over longer ones. PacBio has improved the process over the years, but input DNA requirements are still above nanogram levels.

Wanunu's team has come up with an approach that uses NZMWs — essentially a nanopore at the bottom of every ZMW — and applies a voltage to drive DNA through the pore. Such a conformation enables picogram amounts of input DNA and also preferentially loads longer DNA fragments, Wanunu said.

As previously described, the researchers build waveguides on a silicon oxide membrane with nanopores that are 3 to 5 nanmoeters in diameter fabricated at the base of the waveguides using a transmission electron microscope.

The team then used an array of six NZMWs and studied how DNA fragments were captured into the NZMWs for fragment sizes ranging from 1 kilobase to 48.5 kilobases.

The team demonstrated that a 10-picogram DNA sample could be loaded in less than one minute. By comparison, the authors wrote, conventional magnetic bead loading of 10-kilobase SMRTbell samples requires 1.5 nanograms of input DNA and takes one hour.

Next, the team demonstrated that they could capture DNA bound to a polymerase to enable sequencing. In PacBio sequencing, the DNA template is bound to a DNA polymerase-streptavidin fusion protein, which then binds to the biotin groups on the waveguides. The team demonstrated that they could create a similar DNA polymerase-streptavidin complex at the base of the NZMW to capture DNA.

They then created a two-by-two NZMW array on a membrane that had around 100 ZMWs to compare sequencing of the NZMW with the standard ZMW. They first tested a 72-base circular DNA and DNA polymerase template. Template DNA and fluorescently labeled nucleotides were added.

The researchers then applied three 1-second-long voltage pulses, which captured and immobilized the DNA on the NZMWs. The polymerase was then activated, and sequencing began, as seen from the fluorescent bursts of the nucleotides being incorporated. The fluorescent bursts were only seen in the NZMWs and not the ZMWs, since no template DNA was loaded into the standard ZMWs in the short 3-second loading time. But all four NZMWs captured DNA during that time.

Finally, the researchers demonstrated sequencing of a known 20-kilobase SMRTbell sequencing construct. They used less than one nanogram of input DNA and applied a two-second voltage pulse to load the DNA onto the NZMW.

Because the researchers were not using PacBio's actual instrument, Wanunu said the team had to design its own bioinformatics to do base calling. The algorithm resulted in a 67 percent single-read accuracy and read lengths of around 1.6 kilobases.

Wanunu anticipated that performance would be better if the actual commercial instrument were used, as opposed to the Northeastern team's experimental design. "The system we're using is one we've replicated based on the design principals of PacBio," he said. "Our substrate has some inherent noise that's higher than PacBio's."

He said that the next step would be to continue to optimize the design — including scaling up the NZMW design so that each NZMW does not have to be fabricated individually. The team is currently working on a method that involves using a "porous substrate," he said, "as opposed to drilling pores into a substrate."

In addition, Wanunu's team is also collaborating with PacBio to develop techniques for direct RNA sequencing. Last year, the NHGRI awarded his lab $1.7 million to develop a version of the NZMW for direct RNA and DNA sequencing at picogram input levels.

Wanunu said that he planned to focus on direct RNA sequencing using his lab-developed NZMW set-up. "Once we get to the point where we can demonstrate high-quality sequencing of DNA and RNA, then we can think about integration" with PacBio's instrument. "We still have a ways to go," he said.