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NASA Sequences DNA in Space for First Time Using MinIon on International Space Station

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NEW YORK (GenomeWeb) – NASA scientists and astronauts and their collaborators have sequenced DNA in space for the first time, marking what some call a new era for manned spaceflight.

The agency sent an Oxford Nanopore MinIon to the International Space Station (ISS) in July, with the goal of determining whether the device could be reliably used in a microgravity environment. As Johnson Space Center microbiologist Sarah Wallace told GenomeWeb at the time, the first step was to learn about the MinIon's fluid dynamics in space. There was a concern that bubbles could form when loading the samples into the device aboard the ISS.

Now, NASA has its answer. "It worked beyond our wildest expectations," Weill Cornell Medicine Associate Professor Christopher Mason told GenomeWeb. "The results showed that sequencing in microgravity on the space station works just as well as it would on Earth. The nanopores work quite happily in zero gravity in processing DNA through the pores. And this really opens up a new era of diagnostics on the space station and for future space missions, for everything from genetics to epigenetics to exobiology. It's really the dawn of a new era in space genomics."

The team's paper, released as a preprint on BioRxiv this week, details its efforts to sequence samples containing mixtures of genomic DNA extracted from bacteriophage lambda, Escherichia coli, and mouse. The experiments generated more than 80,000 reads with mean 2D accuracies of 85 percent to 90 percent, mean 1D accuracies of 75 percent to 80 percent, and median read lengths of approximately 6,000 bases. In addition, the researchers were able to generate directed assemblies of the E. coli, lambda, and mouse mitochondrial genomes, and de novo assemblies of lambda and E. coli genomes.

The team further benchmarked these results against MinIon sequences of E. coli, lambda, and mouse DNA generated on Earth, as well as sequences generated on the Illumina MiSeq and PacBio RS II platforms. "The cornerstone of most good science is to have orthogonal validation," Mason said, explaining the need to compare the space-generated sequences against additional technologies. "We ordered very standard aliquots of mouse, E. coli, and lambda DNA, but there are always variations between batches and extraction methods, so we wanted to make sure that when we compared the accuracy of the sequencer or the methods we were seeing on Earth versus in space, that we had really well-validated references of the exact same material we used, orthogonally validated on multiple platforms."

In its study, the team noted that "no observable decrease in MinIon performance was observed while sequencing DNA in space," across all surveyed metrics.

Some of the challenges the team anticipated running into were also less problematic than predicted. For example, NASA research engineer Kristen John told GenomeWeb that the team was concerned about vibrations from the launch and how that might affect the flow cells. But the challenges of vibrations and the possibility of bubble formation were overcome, she said.

Further, added NASA microbiologist Sarah Castro-Wallace, the team was able to maintain the flow cells at a consistent temperature throughout launch, which kept them viable. And now that a procedure has been established, she said, the consistency should be easy to maintain in future flights.

Now, the plan is to test how far the flow cells can be pushed in order to determine their limits aboard the ISS. "We're really going to test the limits of the hardware, and we're going to do some runs later on in the coming months to see really what its life span is up there," Wallace said, "and to see if we see any changes as compared to flow cells that were manufactured from the same lot that are sitting in refrigerators here on the ground."

It's Come a Long Way

The idea to sequence DNA in space using the MinIon took a lot of time and effort to bring to fruition, but it started with a fast experiment.

Researchers had met to discuss the NASA Twins Study — an experiment to study the Kelly brothers after astronaut Scott Kelly spent a year on the ISS while his twin, retired astronaut Mark Kelly, was on Earth — and the conversation turned to sequencing.

It was decided that the sequencer would be sent on a parabolic flight — what some NASA veterans call the "Vomit Comet." A reduced-gravity aircraft follows a parabolic flight path and then freefalls in a series of controlled dives that simulate short, 30-second bursts of microgravity. "This was kind of a late-breaking opportunity to get on the flight, and we were looking at it as a way to test all of our procedures. Can a person actually load a sample in microgravity?" Aaron Burton — a NASA planetary scientist and senior author on the new paper — told GenomeWeb.

On its face, the results of that experiment — which were released in a preprint on BioRxiv in December and which should be published soon — could be considered a failure. The researchers generated only three reads with changed error profiles.

However, Burton said he prefers to think of it as a successful proof-of-principle. The idea was less to get a high-quality sequence than to determine whether the idea could become a reality. "We sent up pre-prepared samples that had been frozen. So there was a lot of stuff we did that the [sequencer] manufacturer would say not to do," he added. "We didn't do any sort of flush step, and also the flow cell was about 12 weeks old, where the manufacturer recommends using them in eight weeks. So we actually looked at that experiment as a success because it showed that a lot of our procedures worked."

Mason agreed with Burton's assessment. "We know definitively from that experiment that it was possible to sequence in microgravity, and that was the first time that had been shown," he said. "What we didn't know is essentially how well it would work. Would it be similar to what we see on Earth or would it be different? Could we sequence and get enough data to do genome assembly in space? We now know the answer is 'yes'."

Step Two

Now that the sequencing has succeeded, the next step is sample and library prep on the ISS.

At the same time they were working on sequencing, the researchers started a separate effort to test the entire process, starting with sample collection and preparation. In July, at the same time a MinIon was winging its way to the ISS, NASA had also sent a device to its Extreme Environment Mission Operations (NEEMO) underwater research station off the coast of Key Largo in Florida. "We had a crew go from DNA extraction from a swab — so nothing culture based, so it was an incredibly low biomass sample — all the way to sequencing on the bottom of the ocean floor," Wallace said.  

The crew tested a sample prep procedure that was spaceflight-compatible, using only what the astronauts aboard the ISS would have at their disposal. It was also done in such a way that it wouldn't necessarily require a trained microbiologist to complete.

"We have a toxicology process that certain items aren't allowed to fly because of their inherent toxicological properties. So we ran all our enzymes and reagents through that first," Wallace said. "No centrifuges — we used things that were already on the ISS or we knew we could easily get certified. So we used a little mini beadbeater that we knew another group had used in the past, we used miniPCR, which already showed that genes can be amplified in space. We used both a thermocycler and a heat block for our enzymes, and we sequenced."

Now, the team is making a push to do sample prep on the ISS itself. Because of the successes at NEEMO, Wallace added, the team is trying to turn that step around quickly.

One thing that could help is another piece of technology Oxford Nanopore is developing — an automated sample prep device called the VolTrax, which should be available in the fall.

"We have asked that we be first on the list to get one when they are ready to start shipping them out," Wallace said. "Oxford has been involved in our NEEMO efforts as well, and they know we're anxious to have an automated system. And I think it's important to note that we're not trying to build an automated system. What we did at NEEMO was manual. We simplified everything as much as possible and came up with a pretty simplistic scheme. But we didn't engineer anything, we didn't develop anything, because we know the company is doing that. We're hoping that Oxford will have that available to us sooner rather than later."

Into the Future

For now, samples sequenced in space will also be sent back to Earth to be sequenced there, a step that will serve as a control. But eventually, the goal is to skip that step and rely on the data generated from the ISS's MinIon. "We could start with some cultures that we're growing in orbit," Wallace said. "We would sequence them in orbit, and then return them to sequence them on the ground just to make sure that everything's matching up. But the goal would be to stop doing that, to reduce sample mass and to be able to make all these calls in near-real time in flight."

Further, Mason added, very soon, ISS researchers should be able to analyze the data in space. "We're tinkering with methods to do real-time analytics for the data, everything from sequencing to base calling to assembly and taxonomic classification all in orbit, and we've shown that it's definitely possible," he said. "As long as we have access to a local compute on the station or a satellite compute, we could do everything off this planet — everything from the chemistry and sequencing to the bioinformatics and classification can all be done in space."

There would be some limitations, he added, simply because of the size of the equipment that the ISS is capable of carrying. "But if you're looking for very specific pathogens or trying to remove host DNA, or even just looking for all bacterial DNA, there's enough there," Mason said. "You couldn't take every existing plant genome and use that as a filter, nor would we need to, so I think with a little bit of hardware improvements, we could get pretty close to putting all of the NCBI database in memory."

And once the researchers can both trust that the data being generated in space is accurate and then analyze it, the possibilities for its use are almost endless.

Initially, the MinIon can be used to sequence samples from both the station and its crew, in order to assess crew health. "I would say it does create this era of in situ diagnostics, and gives you real genetic and molecular diagnostics solutions that can be used quickly," Mason said. "If you have an infection, instead of guessing what it is, you can find out what it is and what are some of the antimicrobial resistance markers present and treat it accordingly. You can do that all in space instead of guessing in orbit and hoping for the best."

As a microbiologist, Wallace said she's "beyond thrilled" at the idea of being able to monitor microbial communities and diagnose infectious diseases in the ISS's air, water, and surfaces in real time, instead of using culture-based methods. "We only have a few media types out there and no incubators, so we're selecting for what we're culturing," she said, "so we're really not getting a complete picture."

Also, being able to correctly diagnose health problems in space could have implications for manned missions to other planets. "It's really important on a long-duration mission where we can't resupply antibiotics or disinfectants to know if it's something worth using an antibiotic or disinfectant on or if it's benign, and those are things we can't yet do," she said.

Further, Johns added, this is the first step to using sequencing on other trips to planets where we might someday go.

"Moving out of low-Earth orbit and planning for 18- to 36-month missions, you're going to need to do in situ diagnostics without returning samples," Burton concurred.

But even more exciting, he added, is the idea of using the sequencer as a "science instrument where you can find interesting samples and do an extraction and see if you find organisms there. So if there's life on Mars, and if it has DNA or RNA as a basis to it, then you can characterize it. If you have something that looks like DNA, but it has a different sugar or you had different bases, you would still be able to measure those things and detect information. That could be one of the most early powerful biomarkers [of non-terrestrial life] you could detect."

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