NASA's Multi-mission Metagenomics Technology Development for Planetary Protection

Since the Viking-era, planetary protection (PP) implementation on missions with biological contamination concerns have been based on values derived from the hardiest known organism, the bacterial spore. While the spore has been a gold standard to assess the biological contamination of spacecraft surfaces, PP requirements have evolved over time to meet the needs of increasingly sophisticated missions. However, advances in nucleic acid technologies have not yet found their way into standard PP practice except as research tools; and there is currently an unmet desire to use these technologies to understand more about the potential "passenger list" for specific mission types and destinations. The current NASA Standard Assay (NSA) method, designed to assess PP risk for spacecraft bound for Mars, is a culture-based method intended to enable bacterial counts that can form endospores, respire aerobically, are resistant to heat processing, and can be cultivated on Tryptic Soy Agar (TSA) media. This technique does not adequately estimate or capture the complete microbial diversity that can be found on the spacecraft. Metagenomics could serve as another implementation approach to meeting PP requirements; one that does not always utilize the worst-case scenario approach for each piece of hardware, so a refined implementation approach and risk assessment can be determined on a case-by-case basis. Technology advancements in microbiology and molecular biology in the last decade can now be utilized to provide an enhanced genetic resolution on the specific biological contamination that may be present on the spacecraft and semi-quantitatively determine how much may be present. In addition, this approach provides a better understanding of the risk of microbial contamination for the targeted extraterrestrial bodies, with lower uncertainty. The development of metagenomic technologies from the current research-based environmental applications to a mainstream application for space hardware would be a leap in technology advancement for the planetary protection discipline and would benefit all future missions. In 2018 NASA initiated a "multi-mission metagenomics" task to assess the current state-of-the-art sample collection, sample processing, DNA sequencing, and bioinformatics pipelines for low biomass spacecraft hardware and associated surfaces. Over the course of the study, three vendors were independently sub-contracted and evaluated to perform state of the art techniques. Each approach has intrinsic strengths and weaknesses when used alone; however, the final success of the multi-mission metagenomics task resides in the integration of these multiple strategies. As a result, we will report the significant findings of these tasks to include the vendor blind study test setup, novel DNA sequencing optimization techniques, a comparative bioinformatic pipeline analysis, and planetary protection specific functional and taxonomic bioinformatic analysis proposed. In this study, it is concluded that sequences pertaining to PP-related microorganisms are present in extremely low numbers and hence in addition to metagenomic approach, NASA should consider developing assays for these PP relevant microorganisms.