In-space DNA sequencing success looks to increase capability with OA-7

When the SpaceX CRS-9 mission launched to the ISS last July, it took with it a first-of-a-kind experiment to examine the possibility of sequencing DNA in space.  Not only did that experiment prove beyond doubt that sequencing DNA on the Space Station is possible, the scientists behind the experiment now have an opportunity to continue their research with a new round of experiments set to launch on Orbital ATK’s Cygnus OA-7 resupply mission.

Sequencing DNA in space – not just possible, but effective:

Shortly after the experiment lifted off on a Falcon 9 and the CRS-9 Dragon resupply mission last July, two members of the DNA sequencing team sat down with to preview the experiment and talk about what might come to pass with the ground-breaking research.

Now, with the experiment on Station for eight months, those same scientists – Aaron Burton, Principal Investigator, and Kristen John, Deputy Project Manager and Project Engineer – once again sat down with to discuss the results of the experiment and what the future holds for the technology.

“On August 26, we performed our first experiment,” stated Kristen.  “Kate Rubins was the astronaut who did the first experiment.  And it went really well.”

While Dr. Rubins performed the first experiment on the Station, Aaron, Kristen as well as Sarah Stahl and Sarah Wallace – the other members of the science team – were at the Johnson Space Center talking directly to her.

“We could see from the video feed that [the experiment] was working pretty quickly.  That was the first experiment, and we sequenced a mouse DNA, a virus, and a bacteria.

“And then we continued to perform another 8 experiments over the course of several months.”

Under the original plan, only three runs were scheduled – with the remaining six samples sent up as contingency or “just in case” back ups”.

As Kristen noted, however, “After the first few runs went really well and we had the right folks on board, we successfully petitioned that it was worthwhile to do the last six, and they let us do all nine of the samples we had sent up.”

With the ability to run six more samples than originally anticipated, the team took the opportunity to change up certain parameters of the experiment.

“We ended up doing a series of experiments where we basically changed the runtime, where some were six hours long and some of them were 48 hours long,” stated Kristen.  

“And then we spaced them out over time.”

Aaron added to this, noting that one of the experiments reused a flow cell, as well.

“We also did an experiment where we reused the flow cell.  So we loaded a sample on it, we ran it for a while, then loaded a second sample, and then continued to run it just to demonstrate more utility in the flow cells.”

Screen Shot 2016-07-21 at 15.53.54Aaron further stated that “initially, we had no idea how much data we were going to generate.  But we found with our 6 hour runs that we would get about a third of the data that we would get if we did a 48-hour run.”

What the team discovered is that they were able to gather enough data after four sequencing runs to sequence the whole genome of the bacteria.

After all nine runs, they were able to get the whole genome of the virus.

“If you add up all of the bases we sequenced during the whole set of experiments, we got about two and a half million bases on the DNA sequence.

“We were close to having the number of bases for the mouse genome,” stated Aaron.

Originally, the experiment had been carefully planned to coincide with Dr. Rubins’ stay on the Station – as her microbiology background and work to develop the experiment and tools on the ground were instrumental in the initial round of sequencing tests.

In all, Dr. Rubins performed seven of the nine experiment runs before she returned to Earth last October.

As luck would have it, shortly after Dr. Rubins departed the ISS, Dr. Peggy Whitson launched as part of the Expedition 50 crew – providing the sequencing team with yet another astronaut with an extensive background in biochemistry.

“We were lucky that we had Kate Rubins and Peggy Whitson be the astronauts doing it,” stated Kristen, who added that Dr. Whitson performed the last two sequencing runs – the last of which took place in January.

2016-07-22-215922“Ultimately, the idea is that anyone can be able to do this,” said Kristen.  “So it was just the way it worked out with who was [on Station] that we had experts showing that they could do it.”

Regardless, the team is confident – thanks in part to runs of the experiment performed during NEEMO 21 last summer – that other astronauts with non-microbiology/biochemistry backgrounds can perform the experiment.

In terms of the experiments’ effectiveness, while it’s difficult to gauge specifics with only nine sample runs, the experiment was actually found to be 1-2% more effective in terms of sequencing accuracies than the parallel, control experiments performed on the ground at the same time.

“For every sample we ran on the ISS we had a parallel sample on the ground,” noted Aaron.

“And we actually found … that we got, pretty much every time, more data generated from the experiments on the ISS than on the Earth.

“We also found that the sequencing accuracies were a little bit higher on the ISS by 1 or 2% than on the ground.”

While there are numerous variables at play, the team is confident with the first round of sample runs to say that DNA sequencing in space is “absolutely no worse” than on the ground.

“At this point, I feel confident saying it works just as well,” said Aaron.  “I’m not confident enough to say it works better.”

Continuing the research – OA-7 Cygnus to launch new round of experiments/equipment:

When Orbital ATK’s OA-7 Cygnus mission launches to the ISS, it will carry with it – among numerous other payloads – a set of new samples and equipment to permit further DNA sequencing runs that will demonstrate the entire process – not just the sequencing of an already prepared sample.

“Over the next couple months, the next step will be to run through the whole process where a crew member will show that he or she can do the whole thing using a mini-PCR that’s already on Station to prepare the sample, using pipettes to move the sample around, and then finally sequencing the sample in the MinION sequencer,” noted Kristen.

The mini-PCR (Polymerase Chain Reaction) is used, in this case, as a heat block to heat up samples as part of their preparation for sequencing.

Screen Shot 2016-07-21 at 15.48.49The mini-PCR that will be used from the upcoming DNA sequencing experiments is on Station not as part of this experiment but as part of the Genes in Space program, run by Boeing, which gives high school students the opportunity to fly microbiology projects to the ISS.

In addition to the unprepared samples, OA-7 will also deliver two new MinION sequencers and new flow cells.

The original MinION sequencers used for the first nine sample runs will be stored for future return to Earth on a Dragon spacecraft.

If the experiment samples launching on OA-7 prove successful, the team hopes to follow that with a series of samples – each containing various bacterial samples – to demonstrate the technology’s ability to distinguish between various types of bacteria.

Eventually, the team hopes to launch samples that contain a mix of various organisms to be sequenced on orbit with that data then put out into the field for researchers to see if they can identify the organisms in the samples from just the data returned from the in-space DNA sequence.

2016-07-22-220627But the real home run experiment, according to Aaron, is the ability to sequence a truly unknown sample.

“What we’d like to have happen is to actually test a real environmental sample that the crew collects up there,” noted Aaron.  “That’s kind of the home run experiment.

“So if the crew can grow something up on a culture plate, the question is then: can we amplify some DNA from that and sequence it?

“That could allow us to identify in a matter of hours what’s growing up there rather than the time it normally takes to return that sample to Earth and characterize it by traditional methods – which can take months.”

2016-07-22-214423Moreover, the entire process, and the MinION sequencer, specifically, should be able to identify any bacterial or viral life form growing/living on Station or infecting one of the crewmembers.

“In principle, it should be able to identify everything that’s up there,” staid Aaron.  

In the realm of bacteria, this species level identification via the MinION DNA sequence is possible due to the fact that all bacteria have a 16S gene, or rRNA – ribosomal RNA.  

As Aaron related, “There are regions where every bacteria has the same DNA sequence and regions where evolution has allowed mutations to occur.  So by looking at the places where those mutations and differences are, you can actually get species-level identification of the bacteria.

2016-07-22-215831“So if you’re looking for bacteria, you can amplify the whole 16S gene, and by looking at the differences in them, you can identify all the different species of bacteria that are in that sample.

“And that’s absolutely what we want to do because just because you know there’s bacteria in your local water sample, you want to know if it’s actually something that’s going to be harmful to you – do you need to treat it – or is it going to be benign.  Because achieving bacterial sterility is really hard.”

Ultimately, the team’s goal is to make this experiment a permanent platform on the ISS that other researchers – including NASA and the Station’s crew – can use.

Until then, the experiment runs continue – demonstrating, importantly, that anyone can perform this type of sequencing.

(Images: SpaceX, NASA, Nanopore Tech, and L2 artist Nathan Koga – The full gallery of Nathan’s (SpaceX Dragon to MCT, SLS, Commercial Crew and more) L2 images can be *found here*)

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