Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition

Supplemental tables and figures for manuscript titled: "Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition" Abstract NASA has been monitoring the microbial burden of spacecraft since the 1970’s Viking missions. Originally based upon culture-based and then focused 16S sequencing techniques, we have now applied whole metagenomic sequencing of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools. Hierarchical clustering analysis separated vacuum particles from SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particle and SAF tricarboxylic acid cycle and amino acid biosynthesis suggested that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 high quality metagenome assembled genomes were generated and were dominated by Moraxella osloensis or Malassezia restricta. A M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This includes a rigorous quantitative determination of microbial loads, and a qualitative dissection of microbial composition. Genomic assembly led to greater confidence of species identification and their functional roles.