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Article

Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing

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JES Tech, Houston, TX 77058, USA
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UCSC Genomics Institute, University of California, Santa Cruz, CA 95064, USA
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Astronaut Office, NASA Johnson Space Center, Houston, TX 77058, USA
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KBR, Houston, TX 77058, USA
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Project Management and Systems Engineering Division, NASA Johnson Space Center, Houston, TX 77058, USA
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Oxford Nanopore Technologies, New York, NY 10013, USA
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Oxford Nanopore Technologies, Oxford Science Park, Oxford OX4 4DQ, UK
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Astromaterials Research and Exploration Science Division, NASA Johnson Space Center, Houston, TX 77058, USA
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Biomedical Research and Environmental Sciences Division, NASA Johnson Space Center, Houston, TX 77058, USA
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Author to whom correspondence should be addressed.
Genes 2021, 12(1), 106; https://doi.org/10.3390/genes12010106
Received: 15 December 2020 / Revised: 11 January 2021 / Accepted: 14 January 2021 / Published: 16 January 2021
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies’ MinION™ sequencer and miniPCR bio’s miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit. View Full-Text
Keywords: nanopore sequencing; in-situ analysis; field-deployable methods; bacterial identification; spaceflight nanopore sequencing; in-situ analysis; field-deployable methods; bacterial identification; spaceflight
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MDPI and ACS Style

Stahl-Rommel, S.; Jain, M.; Nguyen, H.N.; Arnold, R.R.; Aunon-Chancellor, S.M.; Sharp, G.M.; Castro, C.L.; John, K.K.; Juul, S.; Turner, D.J.; Stoddart, D.; Paten, B.; Akeson, M.; Burton, A.S.; Castro-Wallace, S.L. Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing. Genes 2021, 12, 106. https://doi.org/10.3390/genes12010106

AMA Style

Stahl-Rommel S, Jain M, Nguyen HN, Arnold RR, Aunon-Chancellor SM, Sharp GM, Castro CL, John KK, Juul S, Turner DJ, Stoddart D, Paten B, Akeson M, Burton AS, Castro-Wallace SL. Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing. Genes. 2021; 12(1):106. https://doi.org/10.3390/genes12010106

Chicago/Turabian Style

Stahl-Rommel, Sarah, Miten Jain, Hang N. Nguyen, Richard R. Arnold, Serena M. Aunon-Chancellor, Gretta M. Sharp, Christian L. Castro, Kristen K. John, Sissel Juul, Daniel J. Turner, David Stoddart, Benedict Paten, Mark Akeson, Aaron S. Burton, and Sarah L. Castro-Wallace 2021. "Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing" Genes 12, no. 1: 106. https://doi.org/10.3390/genes12010106

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