BEST Station experiment aims for in-situ bacterial identification, sequence RNA for first time in space

by Chris Gebhardt

When the S.S. J.R. Thompson (OA-9E) Cygnus arrived at the International Space Station last month, it brought with it the Biomolecule Extraction and Sequencing Technology, or BEST, experiment that will aim to demonstrate the ability to perform in-situ identification of microbes onboard the International Space Station, study mutation rates over time in bacterial genomes, and perform the first-ever sequencing of RNA, ribonucleic acid, in space.

Speaking in an exclusive interview to Chris Gebhardt of NASASpaceflight, Principal Investigator for BEST, Dr. Sarah Wallace, and co-investigator Dr. Kristen John related the background and aspirations for the project – which in many ways is a carry-on of capability demonstration from the highly successful Biomolecule Sequencer Project and Genes In Space-3 experiments.

In fact, the core team of four scientists from the Biomolecule Sequencer Project and Genes In Space-3 are the same for the BEST experiment, with Dr. Sarah Wallace serving as Principal Investigator joined by co-investigators Dr. Aaron Burton, Dr. Kristen John, and Sarah Stahl. In addition, for the BEST experiments, Dr. Mark Akeson, Dr. Miten Jain, and Dr. Benedict Paten from the University of California Santa Cruz joined the team, with their expertise is allowing for more science to be completed.

Background – success of DNA sequencing in space:

BEST is – in part – made possible due to the success the core team of four scientists has had in demonstrating the ability to sequence DNA and identify microbial life forms from pre-packaged and on-orbit prepared samples launched to the International Space Station on both SpaceX Dragon and Orbital ATK Cygnus vehicles over the last two years.

“Our first experiment, Biomolecule Sequencer, was really just [looking at] does the sequencer work in space?” related Dr. Wallace.  For the Biomolecule Sequencer Project, the team prepared samples on the ground ahead of time with Dr. Kate Rubins then performing the sequencing runs on Station using the MinION nanopore DNA sequencer.

Dr. Kate Rubins, seen with the in-situ results of one of the first DNA sequencing runs performed in space. (Credit: NASA)

“At the same time we were flying [the Biomolecule Sequencer experiment], our team received funding to develop the sample prep process to go along with the [MinION] sequencer so that we could actually prep samples in space, and that’s what Genes in Space-3 was – to show that we could indeed take a raw sample, either launched from the ground or microbes collected from and cultured on the Station and go all the way from sample to answer with the sequencer in space.”

In this way, each of the experiments, from Biomolecule Sequencer to Genes In Space-3 to BEST build upon the previous, with each experiment introducing more capabilities to demonstrate what the sequencing technology can do in space.

The Biomolecule Sequencer Project proved in 2016 that sequencing DNA was indeed possible in space – and is actually better than or the same as DNA sequencing performed on the ground.  “The simple answer is that everything was better than or equal to what we see on the ground in terms of everything, including error rate, the speed at which the molecules go through the nanopores, the quality scores,” related Dr. Wallace.

“Any kind of metric that we could look at, it was equal to or better than Earth.  And so that was really exciting because we found that there was no negative effect of sequencing in space, and we found that we were always getting more data from the spaceflight runs.  And that carries through to today.”

The fact that the team was able to get more data in the DNA sequencing performed in space raised all sorts of questions and proposed hypotheses as to why space-based DNA sequencing provided more information than ground-based sequencing.  Nevertheless, subsequent DNA sequencing runs on the Station have shown that this greater data collection is not a fluke but in fact the apparent baseline – as each DNA sequencing run since the initial test has returned more data than its ground-based comparison runs.

This additional data was measured by the number of DNA molecules “read” as the DNA passed through the MinION nanopore sequencer.  “Whenever a molecule of DNA is sequenced in a nanopore, we call that a read. And so one thing you look at is how many reads you’re getting.  And we got more reads of DNA in space.

“And so within the first few [runs, you’re asking,] is it a coincidence?  But at this point, we’ve done 12 or 13 sequencing runs in space, and every single time we have gotten more DNA sequenced in space than [we have with the samples on the ground].

There’s no concrete understanding yet for why this is so, with Dr. Wallace noting, “At this point [we just have theories], but it’s exciting, nonetheless, that it definitely works as well or, you can even argue maybe better, in space.

BEST – three experiments in one:

Overall, BEST is composed of three different experiments designed to demonstrate the capabilities of the sequencing process and hardware in space and the science that can be performed in-situ on the Station.


The first element of the BEST experiment is what’s known as swab-to-sequencer, an experiment that involves the Station crew not only preparing samples for sequencing but taking those samples directly from surfaces of the International Space Station.  This deviates from the previous elements of Genes In Space-3, as those microbial samples were cultured after collection on Station. For BEST, the team has removed the need to culture the organisms and will sequence directly from the swab.

A swab from the swab-to-sequencer experiment being transferred into miniPCR sample tubes. DNA from the swab will be collected and then the gene providing the bacterial identification amplified with miniPCR. (Credit: NASA)

In this way, it is hoped that the swab-to-sequencer part of BEST will allow for a more complete picture of the microbial environment aboard the International Space Station.  Currently, astronauts swab surfaces and then prepare petri dishes where those microbes grow in colonies on the dish – colonies that are then analyzed on the ground.

The problem there is that not all microbes grow on the petri dish environment available – an environment restricted in some respects for crew health and safety.  This means that the complete microbial picture of the International Space Station is incomplete based on currently available practices.

The BEST swab-to-sequencer experiment might change that.  With this experiment, “we hope to get a more complete understanding of the microbes there,” noted Dr. Wallace.  “Right now, when [an astronaut] swabs a surface, only a small percentage of the microbes that are on that surface can actually grow under the conditions we provide.  So when we get a micro-snapshot of what’s there, it’s not really complete. It’s only what was there that would grow on the certain type of media, at the certain temperatures, with the certain oxygen level, and all of those things” that we can see.

While the current picture is incomplete, it is still more than acceptable for crew health and safety because, as Dr. Wallace states, “most all the pathogens we’re really concerned about will grow under those conditions.”

Now, BEST will aim to provide a complete picture of every microbial lifeform on the Station because the swab-to-sequencer experiment removes the need to culture the sample before identification.  “This will show us everything that’s there because we don’t need to culture it,” said Dr. Wallace.

The Biomolecule Sequencer (Credit: NASA)

This type of swab-to-sequencer identification technique – if successful – could greatly help maintain and even increase crew health and safety standards because by removing the need to culture a sample, the team is eliminating a potential scenario where a dangerous microbial cell would be multiplied into the trillions by culturing it before identification.

While the experiments this time will only look at microbial identification from surfaces on the Station, future applications could be used to identify the microbial environment in an astronaut’s wound during ISS expeditions or deep space exploration missions beyond Low Earth Orbit.

For the experiment itself, the S.S. J.R. Thompson Cygnus delivered 200 swabs that will be used to collect samples for sequencing – with multiple swabs being used to collect samples from the same surface areas.  “We’re hoping that we get a least 20 or so swab sets back from different locations,” noted Dr. Wallace. While some of the swabs will be sequenced onboard the Station, others will be frozen for return to Earth on an upcoming SpaceX Dragon mission, after which the team will retrieve the swabs and sequence them in the lab for comparison with the on-orbit sequencing runs.

For Dr. Wallace, this is the part of BEST she’s most excited about.  “As a microbiologist, we’ve been doing microbiology in space the same way really since Apollo.  And that is great, it’s worked, it’s perfect. We’ve got a healthy crew. We’ve got the vehicle clean.  Everything’s been great.

Dr. Peggy Whitson performs a biology experiment aboard the International Space Station. (Credit: NASA)

“But this is really exciting because it’s taking us to kind of the next level, to where we could really envision getting an answer in a matter of hours versus literally weeks until we get the sample back in the lab.  So for me, it’s definitely seeing that kind of shift in how we view microbial monitoring in the future” that’s really exciting.

Part 2: RNA sequencing:

The second part of BEST will include a never-before-attempted experiment in space: sequencing of RNA.  While DNA has already been successfully sequenced multiple times on orbit, the sequencing of RNA has not, and as Dr. Wallace relates, directly sequencing RNA can provide insights that sequencing of DNA cannot.

“RNA and DNA give you two different things.  DNA is kind of the blueprint of all the potential things your cells can do.  Your genome is capable of so many things (what DNA allows), but you don’t always need all of those things at once,” relates Dr. Wallace.

In simple terms, your RNA is the commander, informing your cell to “do this” and “don’t do that.”  Once your RNA gives an instruction, the RNA then derives the proteins which actually carry out the command.  In essence, RNA tells you what genes are “on” and what genes are “off”.

If a microbiologist needs to know what bacteria is growing on a specific surface of the ISS, they need only sequence its DNA because DNA will tell them who that particular bacteria is.  But if you want to know how that specific bacteria – or any living thing – is responding to spaceflight, DNA is not what you want.

In that case, RNA is what you want to sequence because sequencing RNA will show you which genes are being turned on and which are being turned off as a response to spaceflight, thus allowing microbiologists to begin to understand how living things adapt to the microgravity and radiation environments (among others) of space.

To this end, the same MinION nanopore sequencer used for the DNA experiment is also the only platform that can directly sequence RNA.  And since the MinION is already onboard the International Space Station, utilizing it for RNA sequencing was a logical next step in the technology demonstration process.

However, sequencing of RNA was not always possible.  “Prior to being able to sequence RNA directly, we first had to convert it to cDNA (copy DNA),” noted Dr. Wallace.  The ability to convert RNA to cDNA was originally thought impossible, as scientists believed that while DNA could give rise to RNA, RNA could not be converted back to DNA.  That all changed years ago with the discovery of an RNA virus and a specific enzyme within the virus called reverse transcriptase.

The reverse transcriptase enzyme is what allows RNA to turn back into DNA.  “Forever, the kind of central dogma was that DNA only goes to RNA. But when we found these RNA viruses and learned how they were [turning RNA to DNA], that’s when we realized that we could take that enzyme and turn RNA from research samples back into DNA so that the sequencers could sequence it,” noted Dr. Wallace.

miniPCR and the MinION, two key pieces of hardware used by BEST. miniPCR amplifies DNA and can be used in any reaaction where a specific temperature is required for optimal enzymatic function, whereas the MinION (Biomolecule Sequencer) sequences DNA and RNA. (Credit: NASA)

But this type of transcription, while effective, can lead to biases in the samples – showing results that aren’t actually there.  “So it’s best if you can just look at it in its raw form, directly, via RNA sequencing,” stated Dr. Wallace. This type of direct RNA sequencing – which is now possible due to advancements in sequencing technology and RNA processing prior to sequencing – allows scientists to gain direct understanding of gene expression from the very small percentage of RNA (1% to about  5%) needed to gain knowledge of how an organism responds and changes at the molecular level to spaceflight.

However, just because direct RNA sequencing is possible in general, there are still times when scientists would want to convert that RNA into cDNA – either to amplify what’s there (part of the nature of cDNA) or to perform comparison cDNA sequencing studies to allow for a more complete picture of the organism being examined.

This RNA to cDNA comparison will be part of the BEST RNA sequencing experiment – with both sequencing runs using ground prepared samples that will be run through the MinION handheld device on the Station.  Performing the sequencing of RNA will – from an astronaut’s perspective – be quite similar to the DNA sequencing experiment performed during the Biomolecular Sequencer Project two years ago.

However, a huge question of whether or not RNA sequencing is even possible in space stems from the fact that RNA degrades extremely quickly thanks to how our RNA systems have evolved.  In short, as soon as our cells produce RNA, our body recognizes the RNA as an invading force and sets out to destroy it. This destruction is accomplished through the production of RNases (Ribonucleases) which “chew away” at the RNA.  Moreover, RNA is single stranded and less stable – in general – than DNA.

In ground laboratories, the delicate nature of RNA and its rapid degradation can be stemmed somewhat by trying to remove the RNases and by wiping down and sterilizing the equipment and tools used for preparation and sequencing.  However, many of these processes for ensuring RNA stability on the ground cannot be used on the Space Station because of the risk that those elements would interfere with the Station’s life support systems.

RNA comparison to DNA. (Credit: chemical structures of nucleobases by Roland1952)

To this end, for the first few runs of the BEST RNA sequencing experiment, Dr. Wallace and her team are looking at the RNA sequencing quality from pre-prepared pristine RNA samples.  Following those runs, the team will assess the crew’s ability to manipulate and prepare RNA for sequencing on the ISS.

Part 3: microbial evolution/mutation over time:

The final element of BEST will be to study the mutation rate of a bacterial genome overtime in the space environment.   “We’ve sent up a bacteria that is a very safe one to work with,” said Dr. Wallace. “It’s [a bacteria that’s] in the water you and I drink all the time.  It’s in the water the astronauts drink. So we’ve sent it up there, and we’re going to have the astronauts culture it.

“After about a week or so, they’re going to take some of that culture and transfer it to fresh media to allow additional generations to grow.  And they’ll keep repeating that so that we get this kind of long-term growth profile of this organism to where we end up with cells that have experienced spaceflight for a long time.”

The experiment will be performed on the ground, too, as a control.  At various points, teams on the ground as well as astronauts aboard the Station will select some of the cells and perform a complete genomic sequencing so that a genetic-level understanding of mutation to spaceflight can hopefully be determined for this particular bacteria.

Crew health on the Space Station and on future beyond Low Earth Orbit mission could be greatly aided by the three-part BEST investigation. (Credit: NASA)

“That sounds pretty simple, like, ‘oh yeah, it’s probably been done before,’ but actually it really hasn’t,” related Dr. Wallace.  “So this will be a controlled look at how mutations at the level of the genome happen and how the spaceflight environment is triggering mutations over time.”

Challenges addressed to certify BEST for Station:

As with all experiments launched the International Space Station, BEST had to meet a strict series of criteria so that it would not adversely interfere with the Station’s operation.  “The interesting thing we dealt with for BEST was certifying this thing called the magnetic bead stand,” related Dr. Kristen John. The magnetic bead stand is what holds the samples and is part of the process of actually preparing DNA for sequencing.

“There’s many, many steps, but one of them in particular is cleaning up the DNA.  And so actually just to get that magnetic bead stand certified, it actually turned out they were quite strong magnets, and so that was kind of an interesting opportunity to go through the Safety Panel,” said Dr. John.  “And then we actually had to work with the Electromagnetic Panel to show that launching this magnet was safe to do.”

Moreover, the swab-to-sequencer portion of the experiment and all of its steps also required certification and practice on the ground before being approved for execution on the Station, a process that saw the experiment performed during the NEEMO-22 underwater expedition off the coast of Florida.

Project scientist Sarah Stahl works with a solution of magnetic particles and the magnetic bead stand. DNA or RNA can be bound to the particles and then collected on the side of the tube with magnetic bead stand, allowing the nucleic acid to be washed and all other components removed. (Credit: NASA)

“For swab-to-sequencer, getting all of that organized and the amount of time we spent understanding what things were needed to make it all happen and to simplify the process so that what would normally take hours in the lab can be done in much less time was fascinating,” said Dr. John.

Part of that certification and simplification process involved testing the swab-to-sequencer experiment on the NEEMO-22 expedition in June 2017, using astronauts who were not experts in microbiology to perform the experiment – thus ensuring anyone could carry out the process onboard the Station.  “By running [swab-to-sequencer] through the NEEMO process, we’ve actually proven that researchers, scientists, engineers, whatever kind of background astronauts might have, they can all run through this process,” related Dr. John.

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