Redefining Spaceflight Microbiology: The Evolution of In Situ Nanopore Sequencing for Microbial Monitoring of Crewed Spacecraft

Microbial monitoring onboard the International Space Station (ISS) is essential to assess risks to both spacecraft and crew. Since human occupation began, onboard microbial culture followed by ground-based analysis has provided data descriptive of a human-occupied environment with fluctuations associated with crew turnover and process escapes from the Environmental Control and Life Support Systems (ECLSS). While this culture-based process has served as the gold standard to alert NASA to anomalies and to provide confidence in the operational controls, the data are biased towards organisms that can be cultured. In 2017, the paradigm that samples had to be returned to Earth for analysis was shifted when unknown microbes, collected and cultured from ISS surfaces, were identified onboard through the use of nanopore sequencing. The following year, culture was removed from the process, and a direct swab-to-sequencer, culture-independent method was validated onboard the ISS. Based on the success of these payloads, the Crew Health Care Systems (CHeCS) BioMole Facility was established. BioMole consists of the hardware, consumables, and procedures needed to support sample preparation and nanopore sequencing. Under the BioMole umbrella, the swab-to-sequencer method and a parallel approach for water analysis has been validated, and a full sample-to-answer process has been demonstrated with the inclusion of onboard data analytics. On the ground, fungal identification and air analysis have also been validated. Just as nanopore sequencing has resulted in the evolution of space-based microbial monitoring, improvements in the technology continue to provide opportunities for further expansion of sequencing capabilities. While previous work (since 2017) has focused on targeted sequencing, metagenomics has emerged as the next phase in environmental sample monitoring using nanopore sequencing. A review of nanopore sequencing for microbial monitoring, as well as the benefits and limitations of metagenomic sequencing, will be discussed here.