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Liquid Biopsy in the Field: Oxford Nanopore’s Chemistry, Hardware Plans

By Allison Proffitt

December 6, 2021 | At Oxford Nanopore’s virtual Community Meeting this week, CTO Clive Brown talked about several company developments, several of which had strong diagnostic components. Of note, he teased the Apple iPad Pro-powered Mk1D with an integrated MinION, and the diagnostic capacities of “outy” sequencing.

Integrated Tablet for In-the-Field Sequencing

In a base calling update that Brown called “massive”, he presented the Bonito base caller online. “I’m interested in going back to running things in browsers,” he said. “The latest browsers can access the GPU on your computer; they’re getting quite powerful.” With just a modern browser and a GPU, users can drag and drop .fast5 files into Web Bonito, and the base calling will be done locally. Brown says it works quite nicely on his iPad Pro. “We don’t need to install drivers; we don’t need to install libraries for GPUs.”

The vision for this type of browser-based base calling is to get sequencing and analysis into the field for real-time diagnostic applications—including everything from sample acquisition to sequencing and analysis.  

Brown presented the MinION Mk1D—combining a MinION sequencing device with an Apple iPad Pro. Calling it the next generation of portable sequencing, Brown highlighted that the Mk1D has no cables, runs off battery, and can use 5G mobile capabilities globally. In the promotional images, the iPad Pro case includes two wells above the keyboard for additional devices. One will be for a MinION, the second could be another MinION or perhaps a sample prep device derived from VolTRAX.

Brown boasted of the power the iPad Pro offered: “Most importantly, that web-based Bonito—the experimental version—we’ve been able to run it on [Apple’s] M1 processors and other versions of the base callers. And M1 is Apple’s new silicon which actually has the GPU and other neural network things on the processor. They’ve integrated it with the processor. We’re getting ‘keep-up’ base calling on the iPad Pro—that’s 100 kb/s. On the M1-Max, we’re getting close to P2-level performance.”

His engineers predict late 2022 for the Mk1D, but Brown said he has other ideas. “I’d like to do this much more quickly.”

The Outy Option

Another development with diagnostic implications is an old sequencing chemistry that Brown hopes to revisit. Brown described two of the three options for moving DNA or RNA through a pore: using an enzyme motor to either drive it in (“inny”) or pull it out (“outy”). In the early days of the platform, Brown said, “I settled on the inny scheme, because my belief was it would be easier to implement with double-stranded DNA… It felt more like it was going to be high-throughput… But it’s been on my mind for a long time to circle back to outy. Some of our earliest and best data was done using outy.”

Brown reported current outy sequencing running at about 200 bases per second, and—because the enzyme motor does not immediately detach—a single strand can be re-read again and again. Even with “fairly naïve software,” and a “fairly primitive pore that isn’t optimized,” the average base calls from this cyclical reading are reaching very high accuracy consensus on a single molecule. “Because this is under software control, largely, you have real-time control” over when to repeat a read and when to release the sample, he said.

What really matters, Brown emphasized, is the “ability to definitively call a mutation or a modification at very high confidence.” The outy sequencing mode will enable adaptive sampling—base calling and aligning the first hundred bases of a sample and choosing to either keep or reject the strand based on the findings. “What I can do is, I can fish for interesting molecules in one step,” he said. Then for interesting molecules, the outy sequencing enables “adaptive accuracy”: “Once we’ve found one, we can hold it on the pore. We can accumulate interesting molecules on the pore.”

Paired with liquid biopsy workflows in the lab or in the field, the outy sequencing mode could potentially enable easy-to-use remote “liquid biopsy” sequencing. “It’s the essence of looking at fragments of blood; it’s the essence of looking at fragments of river, it’s the essence of looking at viruses in air droplets,” Brown said, “Anywhere where you get a heterogenous mixture of fragments and you’re looking for a subset with high confidence.”

The hardware for this application stays the same, though the software, front-end, and kit will need to be customized. The company is targeting mid-2022 for a demonstration.         

Enabling the work without a lab at all is Brown’s goal. “I intend to make a liquid biopsy platform,” he said, showing prototypes that use a swab input and are compatible with the MinION platform. Small molecule sensing will be feasible, Brown said, as well as cell-free DNA. “There’s still a lot of work to do on these; it’s very early days. But I’m much happier with the direction it’s going in.” 


For a full summary of Brown’s presentation, see the story at Bio-IT World.

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