SINCE THE BEGINNING OF SPACE EXPLORATION THERE IS A CONCERN ABOUT INTERPLANETARY CROSS-CONTAMINATION. ROBOTIC VEHICLES AND THE EXTERIORS OF MANNED SPACECRAFT REPRESENT EXTREME ENVIRONMENTS FOR EARTH ORGANISMS DUE TO THE PHYSICAL EXTREMES OF OUTER SPACE AND THE THOROUGH DECONTAMINATION PROCEDURES APPLIED TO SPACECRAFT BEFORE LAUNCH. HOWEVER, RECENT STUDIES SHOW THAT MAMY MICROORGANISMS ARE ABLE TO WITHSTAND THESE HARSH ENVIRONMENTS/PROCEDURES AND EVEN SURVIVE UNDER MARS-LIKE CONDITIONS. CURRENT NASA STANDARD PROCEDURES FOR ASSESSING MICROBIAL BIOBURDEN ON SPACECRAFT INVOLVE CULTURE BASED METHODOLOGIES THAT ARE TIME CONSUMING. ALSO, A SERIOUS LIMITATION OF THIS APPROACH IS THAT MOST MICROBIAL ORGANISMS ARE NON-CULTIVABLE AND HENCE, IT IS LIKELY TO SEVERELY UNDERESTIMATE THE LEVEL OF MICROBIAL CONTAMINATION. HERE WE PROPOSE TO DEVELOP AN EFFICIENT AND SENSITIVE MOLECULAR METHODOLOGY FOR THE RAPID IDENTIFICATION AND QUANTIFICATION OF CONTAMINATING MICROBIOTA BASED ON LAMP AMPLIFICATION AND MINION SEQUENCING. TO ACHIEVE THIS GOAL, WE PROPOSE THE FOLLOWING SPECIFIC AIMS: AIM 1: QUANTITATIVE AND QUALITATIVE ANALYSIS OF MICROBIAL SPECIES PRESENT ON THE SURFACE OF CLEANROOMS BEFORE, DURING AND AFTER SPACECRAFT ASSEMBLY IN PREPARATION FOR THE INSIGHT MISSION. WE WILL PERFORM BACTERIAL 16S/FUNGAL ITS1 AMPLIFICATION BY QPCR FOLLOWED BY ILLUMINA SEQUENCING OF SAMPLES COLLECTED FROM CLEANROOM SURFACES AT KEY TIME-POINTS DURING MISSION PREPARATION. QUALITATIVE AND QUANTITATIVE COMPARISON OF MICROBIAL SPECIES PROFILES BEFORE AND AFTER PMA TREATMENT WILL ALLOW US TO CONSTRUCT A CATALOG OF ORGANISMS ABLE TO ENDURE THE DECONTAMINATION TREATMENT APPLIED TO SPACECRAFT AND CLEANROOM SURFACES. AIM 2: GENOMIC CHARACTERIZATION OF MICROBIAL COMMUNITIES THAT WITHSTAND SPACECRAFT DECONTAMINATION PROCESSES AND ASSESS THEIR CAPACITY TO ENDURE OUTER SPACE OR EXTRATERRESTRIAL ENVIRONMENTS. WE WILL IDENTIFY GENETIC SIGNATURES SPECIFIC OF MICROORGANISM RESISTANT TO DECONTAMINATION AND/OR EXTRATERRESTRIAL ENVIRONMENTS THROUGH WHOLE GENOME SEQUENCING AND ANALYSIS OF 180 BACTERIAL ISOLATES THAT HAVE BEEN SHOWN TO SURVIVE IN CONDITIONS SIMILAR TO THOSE FOUND ON THE MARTIAN SURFACE. IN ADDITION, WE WILL PERFORM A METAGENOMIC SEQUENCING APPROACH TO CHARACTERIZE THE METABOLIC CAPACITY OF THE MICROBIAL COMMUNITIES PRESENT IN THE INSIGHT SAMPLES AND EVALUATE THEIR POTENTIAL OF SURVIVING ON THE MARS SURFACE BY COMPARATIVE ANALYSIS AGAINST METAGENOMIC SAMPLES FROM RELEVANT EXTREME ENVIRONMENTS AND WHOLE GENOME SEQUENCING DATA FROM THE 180 RESISTANT ISOLATES. AIM 3: TO IMPLEMENT A RAPID AND COST-EFFECTIVE LAMP 16S/ITS1 AMPLIFICATION AND MINION SEQUENCING PROTOCOL FOR THE IDENTIFICATION OF MICROBIAL CONTAMINANTS PRESENT ON THE SURFACE OF ASSEMBLY CLEANROOM. WE WILL EVALUATE THE PERFORMANCE OF MINION TECHNOLOGY DURING THE CHARACTERIZATION OF EXTANT MICROBIAL COMMUNITIES FROM INSIGT SAMPLES COMPARED TO ILLUMINA-BASED TAXONOMIC PROFILES FROM AIM 1. WE WILL ALSO DEVELOP A LAMP PROTOCOL TO AMPLIFYING 16S AND ITS1 MOLECULAR MARKERS THAT WILL THEN BE SEQUENCED WITH MINION. FINALLY, WE WILL ADJUST JCVI 16S/ITS1 ILLUMINA PROCESSING PIPELINE TO WORK ON MINION READS DERIVED FROM LAMP AMPLICONS TO IMPROVE MINION-BASED TAXONOMIC PROFILING. THE PROPOSED RESEARCH IS RELEVANT TO THE PLANETARY PROTECTION RESEARCH PROGRAM IN THAT ITS MAIN GOAL IS TO DEVELOP A NOVEL, SENSITIVE MOLECULAR ANALYTICAL METHODS FOR THE RAPID IDENTIFICATION AND QUANTIFICATION OF A BROAD RANGE OF MICROBIAL ORGANISMS TO ASSESS THE LIKELIHOOD OF FORWARD (AND POTENTIALLY BACKWARD) CONTAMINATION. SIGNIFICANCE: ONCE DEVELOPED, THE PROPOSED METHODOLOGY WILL HELP SETTING NEW, MORE SENSITIVE AND INFORMATIVE STANDARDS FOR ASSESSING BIOBURDEN DURING SPACECRAFT PREPARATION AND OPERATING PROCEDURES ON EARTH, THAT CAN POTENTIALLY BE APPLIED DURING SPACE MISSIONS OR IN OTHER PLANETS.