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Northeastern University Researchers Develop New Type of Zero-Mode Waveguide for Sequencing

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This story has been updated to include additional information about the authorship of the DNA fragments study.

NEW YORK – Researchers from Northeastern University have created a new flavor of zero-mode waveguide (ZMW) sequencing, the technology underlying Pacific Biosciences' long-read platform, which they are planning to explore for RNA sequencing using recent funding from the National Institutes of Health.

Led by Meni Wanunu, a professor of physics, chemistry, and chemical biology at Northeastern, the team has developed electro-optical ZMWs (eZMWs), which add an electrode to the base of the well that makes up each ZMW. This advance helps DNA loading for each feature, removing length bias seen in more vanilla versions of the technology and unlocking the ability to sequence samples with small amounts of DNA — potentially under a picogram.

"One of the challenges of all sequencing methods is, you need to prepare libraries by amplification of DNA," Wanunu said. "Right now, everything is relying on making libraries and adding adapters. We're trying to eliminate the amplification part."

The problem with DNA amplification is that critical information gets lost, "the main one being the ability to look at chemical modifications," he added.

"What they demonstrate is very straightforward," said Dan Branton, professor emeritus at Harvard University and an expert on the use of nanotechnology in sequencing. "Essentially, concentration of DNA leads to much better coverage of the waveguide than would be possible without the electromotive force."

"I think it could be a very valuable addition to the function and utilization of single-molecule, real-time sequencers," he added.

In proof-of-concept work published in December 2021 in Advanced Materials, Wanunu's lab sequenced DNA fragments using its new eZMWs. Molecules ranged in length from 260 bp to 26 kb. But the true power of the technology may be its potential to directly sequence RNA. Wanunu's lab has received $2.2 million in funding to explore this direction, including $858,993 in 2022.

The work builds on the ZMW technology underlying Pacific Biosciences' long-read sequencing method. Wanunu and PacBio CSO Jonas Korlach have coauthored at least one paper, and Korlach was named as a collaborator on a poster at AGBT this year with the same title as the Advanced Materials paper; however, Korlach was not listed as an author on the paper. Wanunu said he offered Korlach authorship on the paper, however, Korlach turned the offer down. PacBio did not not immediately respond to a request for comment. Wanunu and Korlach were also co-PIs on an $825,000 grant from the National Human Genome Research Institute awarded in 2012.

Wanunu said he has "no official relationship with the firm," though PacBio was issued a sub-award on one grant to sequence samples as the Wanunu lab built its devices, and the lab has received in-kind contributions of reagents. But beyond that, PacBio appears to be staying an arm's length away from the eZMW research. "We had to rebuild the PacBio platform in our lab," Wanunu said, resulting in lower accuracy for longer reads. "It's still a thing we're struggling with. A few million dollars is not enough to build the state-of-the-art machine PacBio has," he said. Moreover, PacBio has not made its software available to the lab, which would help with accuracy, he said.

The eZMW work also builds on an attempt to use nanopores to help draw molecules to the waveguide. From 2016 to 2019, the Wanunu lab received approximately $2.3 million in NHGRI funding for "direct picogram DNA and RNA sequencing using nanopore ZMWs." In 2017, Wanunu and Korlach published a paper in Nature Nanotechnology showing preferential loading of longer molecules and lower input requirements.

But the nanopore membrane led to short-circuiting and increased background noise, according to graduate student Fatemeh Farhangdoust, who led the charge to come up with an alternative, an electrode placed a few nanometers underneath the waveguide. "Now this new platform is very stable," she said. "Every chip coming out of the wafers is useable, and it is very easy to scale up."

The team collaborated with the Cornell University NanoScale Science and Technology Facility, which had strontium lithography and a beam evaporator needed to make the eZMWs. Wanunu said he has submitted a patent application on eZMWs.

While last year's paper helped show proof of concept of the technology, "being able to rapidly identify DNA has applications," Farhangdoust noted.

One is looking at an unknown sample. "If you can get a quick readout, you can identify which genomes are present," Wanunu said, especially if you have a metagenomics database to compare sequences to.

In addition, the low input DNA requirements would potentially be helpful for liquid biopsy assays, though the accuracy issues would preclude that application for now. The lab is working to overcome those, Wanunu said, noting that he has hired a software engineer to improve accuracy, aiming for percentages in the high 90s.

Though they haven't demonstrated it yet, at least not publicly, the killer application for eZMWs may be the ability to sequence RNA. "This is where it's heading: single-cell RNA sequencing without having to make libraries and amplicons of the RNA," he said.

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