With the arrival of the SpaceX CRS-9 mission to the Space Station Wednesday morning, the first-ever in-space DNA sequencer experiment has arrived at the international orbiting lab. The experiment is designed to test the feasibility of DNA sequencing in a non-Earth environment as well as serve as a pathfinder for sequencing initiatives on long-duration human missions in the inner solar system and on Mars.
DNA sequencing in space — the process to get to Station:
NASASpaceflight.com’s Chris Gebhardt recently sat down with two members of the Biomolecule Sequencer Project – Aaron Burton, the Principal Investigator and astrobiologist, and Kristen John, Deputy Project Manager and engineer – for an interview about the potentially revolutionary project.
While launched Monday morning to the ISS, work on the project began over four years ago when Aaron was in a Postdoc program at the Goddard Space Flight Center.
“Our research was on astrobiology and trying to understand the origins of life. One of the people there talked to one of the inventors of nanopore sequencing, and they were interested in sending one of their sequencers up as a planetary science instrument to Mars in order to search for DNA-based life,” related Aaron.
When Aaron accepted a job at the Johnson Space Center in 2013, he thought that an initial step for nanopore sequencing in space could be sending a sequencer to the ISS to demonstrate that DNA sequencing was possible in a non-Earth environment.
“At that point, the technology was still in the development phase. So we ended up getting turned down for the technology demonstration because we didn’t have the sequencer in hand,” said Aaron.
As the project continued, NASA astronaut Dr. Kate Rubins became involved, helping the team work through concepts and pre-mission planning activities involving the sample prep process (the samples that would be used to sequence DNA on orbit) and the experiment runs themselves.
Then, in late-December 2014, Oxford University “put out a call to get beta testers for their MinION nanopore DNA sequencer,” noted Aaron.
The team submitted a proposal and were delighted to learn in early 2015 that they were accepted as beta testers and that their desire to send a sequencer to space resonated with the research team in Oxford.
Coincidentally and beneficially, at the same time, engineers at the Johnson Space Center initiated a new certification process for hardware flying to the ISS.
According to Aaron, “Normally flight Hardware is Class 1, and they came up with a new designation called Class 1E – meaning experimental hardware, technology that is not on the critical path for crew or life support.
“If it doesn’t work, people’s lives aren’t in danger.”
This new Class 1E designation, while still adhering to safety requirements for humans and the vehicle itself, allows researchers to assume slightly more risk about whether or not their hardware will work.
That “slightly more risk” accommodation means that teams are not required to do parabolic flight testing or vibration testing on their equipment if they think their technology won’t need that prior to flight.
Moreover, since the hardware itself, the MinION from Oxford, was an off-the-shelf product with nothing that needed to be designed in house, the experiment was officially selected for flight in February 2015, with final hardware delivery in December 2015 after just 10 months of development.
Both Aaron and Kristen noted that the 10 month timeframe would normally have taken three to four years under the Class 1 designation.
However, while the experiment was ready to fly in December 2015, more than enough time to make the April 2016 launch date of SpaceX’s CRS-8 mission, the experiment itself required Cold Storage capability aboard SpaceX’s Dragon capsule.
Moreover, the team was expressly trying to overlap the experiment with Dr. Rubins’ flight.
“In principle, we would like for anyone to be able to do this,” stated Aaron. “If you had a crew member who didn’t have a biology background [like Dr. Rubins does], it might require more hands-on training time with them.
“So it’s not a requirement that Dr. Rubins do the experiment, and we want to make it – especially as we develop procedures to use in-flight down the road – so that anyone can do it. But having Kate for our first try when we’re trying to show that it works at all is incredibly fortunate.”
Also fortunate for the team was the size and weight of the MinION itself and the various frozen samples prepared before flight.
“The MinION is a handheld device that weighs 120 grams (0.264 lbs) and fits in your hand,” noted Aaron. “The next closest sequencer is about the size of a large microwave, and it weighs about 110 pounds.
“If you’re going to do a technology demonstration and you’ve got something like we have, which is two MinIONs, 9 flow cells, 9 samples, some positive displacement pipettes, and a Surface Pro 3, all of that is under 5 pounds – which means it costs significantly less to launch all of that to the Station then it would that microwave sized sequencer.”
That microwave-sized sequencer also uses “conventional sequencing with fluorescently labeled nucleotides (organic molecules – A, G, T, and C – that serve as the monomers, or subunits, of nucleic acids like DNA). And so you’ll have a strand of DNA, and you copy it. When you incorporate a new nucleotide, you get a fluorescent signal that comes off.
“Those fluorescent measurements are how you read conventional sequencing. But that means you also have to have lasers that are very carefully aligned.”
The MinION, on the other hand, operates through electrochemical detection, a much more simple and effective way of sequencing for space-based operations.
“The way the MinION works is that we have these nanopores that are just big enough to allow a single strand of DNA to pass through.
“How we actually sequence DNA using the MinION is that normally there is nothing in the nanopore, and you have a certain amount of ions that are flowing through. That’s our baseline current.
“Then, as DNA passes through the pore, the current changes. And that change in current is diagnostic of the sequence of DNA passing through it.
“The pore is big enough so that five nucleotides fit in there at one time. So if you had five As sitting in there, you would get a characteristic current. And then the next nucleotide is fed through by a protein, pushing the first A out. And let’s say that new nucleotide is a T, then the next current you’re going to measure is the current of four As and one T.
“And that is a different current than the one with 5 As.
“The MinION will then feed through another nucleotide, and you’ll see a different current. So basically you’re measuring how the DNA is blocking the pore, and the current changes depending on which nucleotides are in the pore.”
This is simple, from the science standpoint, and importantly is not the part of the process that the team is worried about in terms of proving that DNA sequencing is possible in space.
According to Aaron and Kristen, one of the primary concerns with this experiment is air bubbles. To this end, there’s an initial step before loading the sample into the machine where the air bubbles have to be extracted before the sample is loaded – all while trying not to introduce any new air bubbles.
On Earth, this is accomplished by putting the sample in a centrifuge, and all the air bubbles rise to the top.
“In space, though, there’s no gravity environment that causes the air bubbles to rise or go anywhere,” noted Aaron. They kind of want to glomb together and make bigger bubbles.
“So our biggest concern is that if you do get an air bubble introduced in the MinION, what’s it going to do? Is it going to stick to a surface? Is it going to interact with the pores? Is it going to block part of the pore?”
The other principal concern that could make the experiment unfeasible in space is the launch vibration environment.
As Aaron related, the samples are “naturally occurring proteins sitting in a membrane. So if you get enough vibration at the right frequency you can actually disrupt those membranes and they can start falling apart.”
“So we actually don’t have a huge question of whether or not the machine, the MinION, will be able to sequence DNA. We’re quite confident on that,” stated Aaron. “It’s the other two things that could hold us up.”
On Station — doing the experiment and getting the data back to Earth
Now that the experiment has arrived on Station, the next step is finalizing the precise time when Dr. Rubens will actually perform the experiment.
While precise dates and times have not been determined (though the samples have to be used no later than 8 weeks after preparation – which occurred just prior to launch in mid-July), the team plans three full sequencing runs, which will require approximately five hours of crew time.
According to Kristen, “Crew time is extremely limited on the Station, so getting five hours of dedicated time is actually a lot and should let us do three complete runs of the experiment.”
In fact, limited crew time was a prime consideration for how this first experiment was designed, with the samples that will be sent through the MinION prepared on the ground before launch.
As Aaron stated, “The process of preparing samples for sequencing takes between three and four hours, and that’s by someone who’s trained and who’s done it many times. And it’s on the ground where you know how things work.
The current NEMO expedition off the coast of Florida as well as researchers at Oxford are working on ways to get this preparation time down to as little as 10mins with non-microbiologists, but for now, it’s a multi-hour process.
“Hopefully, going forward, any astronaut will be able to do this in flight. But for this initial run, we wanted to make sure that the sequencer actually works,” noted Aaron. “So in-flight sample preparation wasn’t a priority.”
Regardless of future sample preparation, the current experiment will specifically test the MinION’s ability to successfully detect and sequence DNA from three different types of creatures: a virus, a bacterium, and a mammal.
The three specific DNA samples are all from viruses, bacteria, and mammals that have well-known, understood, and characterized DNA – providing a clear baseline for each DNA sequence in the experiment.
As Aaron stated, the reason for such a diverse test is because “we want to show that you can sequence DNA from basically anything, whether it’s a virus or an entire organism.”
For the actual experiment, Aaron and Kristen noted that there’s a total of about 100 nanograms of DNA in the samples, and they’re hoping to get somewhere around 10,000 sequence reads from the MinION on the samples.
“We’ve done our best to ensure that there’s an equal number of virus, bacteria, and mammal in the samples. So hopefully we’ll be able to get about 3,000 sequences from each organism,” stated Aaron.
The sequences will be measured in a current vs. time plot because the MinION measures the change in current over a length of time that nucleotides are blocking the pore entrance.
Because of the fast-paced nature of the sequencing process, the sequencer will be hooked up to a USB 3.0 connection that will tie the sequencer to a solid state recorder in the Surface Pro 3, where all of the data will be recorded locally before it’s transmitted in its raw form to the ground for analysis.
In-space DNA sequencing – a tool for astrobiology with Earth-based benefits:
As with many, if not all of the experiments performed on the ISS, this in-space DNA sequencing experiment holds potentially enormous benefits for future exploration of the inner solar system in terms of astrobiology as it also does for real-world applications on Earth.
For those real-world applications, Aaron noted that “In many ways, the ISS is analogous to how it is in third world countries. If you’re out in Africa studying the recent Ebola outbreak, which people were actually doing with the MinION, you need to have portable diagnostic capability in the field, and you need the machine to not require a lot of resources.
“Anything we can do to make this technology usable by anyone, not just trained microbiologists, means that a non-specialist doing field work on water monitoring in a resource limited place on Earth would be able to use it.”
In terms of astrobiology, Aaron stated, “There have been a lot of cases made for why both Mars and Earth might have DNA-based life. Some of the basic ones are that we have Martian meteorites here on Earth, so we know that material was exchanged between the planets.
“At some point in its past, Mars probably had more of an atmosphere and had liquid water. So it had a similar sort of environmental context. So if an organism had traveled from one planet to the other, common conditions might not have been so dissimilar that life would have necessarily died.
“And so if you did have DNA-based life on Mars, the MinION could help us detect that without having to replicate the DNA as other sequencers do.”
Moreover, if life – even fossilized life – were to be found on Mars, it’s entirely possible that all other sequencers would not be able to sequence that life’s DNA because of those sequencers’ need to copy the DNA itself.
“Most of the sequencing technologies require you to copy the DNA you have,” states Aaron. “We have a lot of really good methods for copying DNA using biological enzymes. But these enzymes are highly evolved to only recognize the A, G, T, and C nucleotides of DNA.
“So if you had some alien organism that instead of A, G, C, and T had H, I, J, and K – whatever the letters of that alphabet are – our enzymes here on Earth would not recognize those. So you wouldn’t be able to do any type of sequencing that was dependent on copying the DNA.
“Whereas with nanopore sequencing, as long as you can feed it through that pore, you’re going to get a current profile vs. time that should be able to see a pattern even if you don’t know what the DNA sequence letters are.
“And that pattern should be something that is not repeating, but not random either. We call that semi-repeating, like our DNA is, and it’s basically saying ‘Hey, there’s some information here.’”
Moreover, Aaron noted that nanopore sequencing could help with in situ investigation of biological components on Mars in terms of making sure that any life we might find there isn’t life we brought with the spacecraft or humans from Earth.
“We do our best to sterilize spacecraft, but even the Curiosity Rover had somewhere around 500,000 spores that were considered viable,” noted Aaron. “We know we’ve brought Earth life to Mars.
“So if we were to find life on Mars, we’d like to be able to know that it’s not something we brought with us on our spacecraft or something that came with the human that was there.”
In terms of more practical applications for astronauts, nanopore sequencing technology like the MinION would also be able to help better diagnose in-flight infections among crew members.
According to Aaron, “Right now, if a crewmember gets an infection, they take a picture or just talk to their flight surgeon and do the best they can.
“But being able to know whether the infection is something that actually requires antibiotics, and if so what kind of antibiotic – if any – is needed for treatment, would really help with diagnostic capability.”
(Images: NASA, Nanopore Tech, Sarah Castro)