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DNA Sequencing in the Final Frontier

10/19/2016
Amber Dance, PhD

The International Space Station entered the genetic age this summer with the first DNA sequencing aboard. Find out about the challenges of sequencing in space...


In microgravity, Kate Rubin's hair floats as she performs the sequencing experiment.

View more images showing preparations for sequencing DNA aboard the ISS.

It was a small step for molecular biology, but could herald a giant leap forward in how astronauts eventually diagnose their own infections or even search for extraterrestrial life. On August 26, 2016, NASA astronaut Kate Rubins thawed a DNA sample and ran it through a sequencer not much bigger than a USB stick. NASA scientists watching a video feed from the ground saw sequences come up in real time. “There were definitely cheers all around the room,” recalled Sarah Wallace, a microbiologist at the Johnson Space Center in Houston. The resulting codes were just as good as those from control sequencing done on Earth, she and her collaborators report in the online preprint server BioRxiv (1).

There’s plenty on the International Space Station (ISS) that Wallace would love to sequence. The station hosts all kinds of microbes: the pathogen Staphylococcus, the water contaminant Burkholderia, the mold Penicillium, and more. These manifest as environmental contamination—such as the funky fungus on some station walls—or infections. For now, Wallace has to wait for cultures to return to Earth to identify them.

While she has never identified anything “super-pathogenic” in ISS environmental samples, Wallace worries that astronauts might bring with them opportunistic microbes, such as Staphylococcus aureus, that could cause disease on a mission. Space travel makes microbes more virulent (2) even as it dampens the human immune system (3). Fast, sequence-based diagnosis could help astronauts identify the right treatment and avoid depleting stores of medications unnecessarily.

Plus, sequencing might help distinguish a microbial E.T. from the Earth organisms that hitch a ride on spacecraft despite NASA’s best efforts to keep the craft clean.

Through the Wormhole

Despite the potential benefits of space sequencing, most modern sequencers aren’t good candidates because they’re big and heavy—about the size of a microwave or mini-fridge. That’s no good when it costs thousands of dollars for every pound sent up to the ISS, said Aaron Burton, a planetary scientist at Johnson Space Center and principal investigator of the study.

Enter the MinION sequencer. “The whole thing weighs something like 87 grams, it’s about the size of a Mars bar,” said co-author Dan Turner of Oxford Nanopore Technologies in the U.K., which makes the device. Scientists have already used it to monitor Ebola evolution in West Africa in 2015 (4).

The MinION works by sending DNA through a tiny pore, a mutant version of an Escherichia coli secretion channel. The pores are mounted in a membrane, and the MinION sends a current through each pore. As the bases squeeze through, each changes the current in a different way, allowing the device to “read” the nucleotides’ identities.

Since the MinION can analyze any polymer that fits through the pores, it’s not limited to DNA. “The thought is that extraterrestrial life would be based on sort of a polymer similar to DNA or RNA,” said Sissel Juul of Oxford Nanopore, co-author of the paper. “If that’s the case, the MinION would certainly be able to detect it.”

Ground Control

To get the most information out of the sequencing runs, the scientists sent syringes pre-loaded with a mix of ready-to-sequence DNA from mouse, E. coli, and bacteriophage lambda. If all went well, the MinION data should be able to identify those three species.

The MinION’s error rate is slightly higher than next-generation sequencing techniques, but it makes up for that with long sequence reads that should provide sufficient data to ID a species, said project collaborator Christopher Mason of Weill Cornell Medicine.

“There was a lot of anxiety,” recalled Mason. “When you’re working with individual, single molecules, they’re very sensitive to degradation or noise in the hardware.” A test-run in parabolic flight to mimic microgravity (i.e. on the “Vomit Comet”) yielded poor sequencing results (5), but Oxford Nanopore has made improvements since then.

One concern was that the cartridges would have to withstand the intense vibration of blastoff, without any disruption to the membrane-bound nanopores. Another was air bubbles. In microgravity, bubbles form easily and are hard to disperse. Air could block the sample loading port of MinION cartridges, so the researchers planned a defensive maneuver. Before she loaded her DNA samples, Rubins used a syringe to suck out a bit of buffer, and any air present, from the port.

Rubins, also a study co-author, is a PhD biologist with a background in infectious disease, so the collaborators were thrilled that she would be able to troubleshoot any problems. As it happened, though, she cruised through each of four sequencing runs, done between August 26 and September 13, with no trouble. Simultaneously, Sarah Stahl at Johnson Space Center ran identical samples through a terrestrial MinION. When the data streamed back to Earth, the authors could quickly compare the two datasets. “The amount of data and the quality of data were comparable,” said Wallace. In fact, the only run to fail was a ground-based control.

The MinION project isn’t the only molecular biology going on in microgravity. One NASA project already completed tested pipetting in parabolic flight (6). PCR was the first molecular biology operation performed in space earlier this year, as part of an experiment designed by 17-year-old Anna-Sophia Boguraev of Bedford, New York, who won the 2015 Genes in Space competition with her proposal. NASA’s Wetlab2 project is also working on DNA and RNA extraction and RT-PCR. Together, these should give NASA tools to better analyze what happens to organisms in space.

Next up, Wallace and colleagues want to make it possible to prepare a DNA sample for sequencing on the ISS, too. Already, the sequencing team has tested a simplified DNA prep protocol in the underwater NASA Extreme Environment Mission Operations (NEEMO) lab off the Florida Keys; the aquanauts swabbed spots in the lab and successfully isolated the genetic material. And Oxford Nanopore Technologies expects to release a sample prep device, called VolTRAX, in December. If the sequencer works in microgravity, then VolTRAX ought to as well, said Turner. The DNA-prep mission could blast off some time in 2017.

Curious about how all of this was accomplished? Take a look at some photographs documenting the process and planning.

References

1. Castro-Wallace, S.L., Chiu, C.Y., John, K.K., Stahl, S.E., Rubins, K.H., McIntyre, A.B.R., Dworkin, J.P., Lupisella, M.L., Smith, D.J., Botkin, D.J., Stephenson, T.A., Juul, S., Turner, D.J., Izquierdo, F., Federman, S., Stryke, D., Somasekar, S., Alexander, N., Yu, G., Mason, C.E., Burton, A.S., 2016. Nanopore DNA sequencing and genome assembly on the International Space Station. BioRxiv, http://dx.doi.org/10.1101/077651

2. Wilson, J.W., Ott., C.M., Höner zu Bentrup, K., Ramamurthy, R., Quick, L., Porwollik, S., Cheng, P., McClelland, M., Tsaprailis, G., Radabaugh, T., Hunt, A., Fernandez, D., Richter, E., Shah, M., Kilcoyne, M., Joshi, L., Nelman-Gonzalez, M., Hing, S., Parra, M., Dumars, P., Norwood, K., Bober, R., Devich, J., Ruggles, A., Goulart, C., Rupert, M., Stodieck, L., Stafford, P., Catella, L., Schurr, M.J., Buchanan, K., Morici, L., McCracken, J., Allen, P., Baker-Coleman, C., Hammond, T., Vogel, J., Nelson, R., Pierson, D.L., Steanyshyn-Piper, H.M., Nickerson, C.A., 2007. Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. Proc Natl Acad Sci U S A, 104(41) http://www.pnas.org/content/104/41/16299.long

3. Taylor, G.R., Konstantinova, I., Sonnenfeld, G., Jennings, R., 1997. Changes in the immune system during and after spaceflight. Adv Space Biol Med, 6 https://www.ncbi.nlm.nih.gov/pubmed/9048132

4. Quick, J., Loman, N.J., Duraffour, D., Simpson, J.T., Severi, E., Cowley, L., Bore, J.A., Koundouno, R., Dudas, G., Mikhail, A., Ouédraogo, N., Afrough, B., Bah, A., Baum, J.H.J., Becker-Ziaja, B., Boettcher, J.P., Cabeza-Cabrerizo, M., Camino-Sánchez, Á., Carter, L.L., Doerrbecker, J., Enkirch, T., Garcia-Dorival, I., Hetzelt, N., Hinzmann, J., Holm, T., et al., 2016. Real-time, portable genome sequencing for Ebola surveillance. Nature, 530 (7589) http://www.nature.com/nature/journal/v530/n7589/full/nature16996.html

5. McIntyre, A.B.R., Rizzardi, L., Yu, A.M., Rosen, G.L., Alexander, N., Botkin, D.J., John, K.K., Castro-Wallace, S.L., Burton, A.S., Feinberg, A., Mason, C.E., 2015. Nanopore sequencing in microgravity. BioRxiv, http://dx.doi.org/10.1101/032342

6. Rizzardi, L.F., Kunz, H., Rubins, K., Chouker, A., Quiriarte, H., Sams, C., Crucian, B.E., Feinberg, A.P., 2016. Evaluation of techniques for performing cellular isolation and preservation during microgravity conditions. NPJ Microgravity, 2 http://www.nature.com/articles/npjmgrav201625