Culture-Independent Fungal Profiling for the International Space Station using Nanopore Sequencing: Method Development

Microbial monitoring of the International Space Station (ISS) environment is a crew health requirement that encompasses both bacterial and fungal identification. Currently, culture-based methods are used for sample collection, and these samples must be returned to the laboratory for analysis. The use of culture and the need for sample return to Earth results in a bias toward culturable organisms and causes a significant delay between sample collection and delivery of final data (weeks to months), respectively. Recently, advancements in molecular technology have aided a broad range of applications, including medical, industrial, and basic sciences. Additionally, increases in portability and ease-of-use of molecular platforms have provided point-of-use capabilities demonstrated by the miniPCR thermal cycler and the MinION sequencer (Oxford Nanopore Technologies). These devices have been applied to, and validated for, the identification of bacteria onboard the ISS. Building on this work, a spaceflight-compatible fungal workflow has developed. Molecular-based fungal analysis is complicated by low biomass, difficult-to-lyse spores, debate regarding the region for taxonomic assignment, and the lack of bioinformatic pipelines and curated-databases. To overcome these difficulties, primers yielding an ~ 2 Kb amplicon were validated against a wide range of fungal isolates. The current spaceflight method was substantially optimized, bioinformatic pipeline was created, and refinements to the UNITE database were implemented. To compare this optimized method to the current culture-based standard, 41 sample sets (82 total swabs, held in tandem) were evaluated. Parallel fungal profiles were obtained between the two methods, with the culture-independent method revealing increased diversity. Further validation has also performed with the launched DNA on board the ISS using the fungal flight compatible method. The addition of this method to the already established bacterial process fulfills the crew health identification requirement. Moreover, the implementation of this method onboard ISS will enhance our understanding of its unique fungal microbiome.