THE OVERALL PROJECT GOAL IS TO PROVIDE THE AGRICULTURAL COMMUNITY WITH A NOVEL, VALIDATED AND HIGHLY DISCRIMINATIVE METHOD FOR TRACKING AND SOURCE ATTRIBUTION OF ANTIMICROBIAL RESISTANCE IN ENVIRONMENTAL SYSTEMS, LEVERAGING NUCLEOTIDE-LEVEL VARIATION WITHIN RESISTOME DATA.THE FOLLOWING OBJECTIVES SUPPORT THIS GOAL: 1) DEVELOPMENT OF COMPUTATIONAL ALGORITHMS AND STATISTICAL METHODS FOR GENERATING NUCLEOTIDE-LEVEL RESISTOME FINGERPRINTS FROM METAGENOMIC DATA; 2) EVALUATION OF THE APPLIED BENEFIT OF RESISTOME FINGERPRINTS UTILIZING BOTH SIMULATED AND PUBLIC METAGENOMIC DATA FROM DISTANTLY AND CLOSELY RELATED SAMPLES; 3) VALIDATION OF THE HIGH DISCRIMINATIVE CAPACITY OF RESISTOME FINGERPRINTS USING EXPERIMENTALLY GENERATED DATA; AND 4) SUSTAINMENT OF THE METHOD VIA AN INTEGRATED OUTREACH CAMPAIGN.TO ACCOMPLISH THESE OBJECTIVES, WE WILL FIRST IDENTIFY AND MODIFY COMPUTATIONAL ALGORITHMS FOR NUCLEOTIDE-LEVEL VARIANT DETECTION IN METAGENOMIC DATA. NEXT, RESISTOME FINGERPRINTS WILL BE GENERATED USING ADVANCED STATISTICAL METHODS COMBINING NUCLEOTIDE VARIANTS, SNP CALLING AND DISTANCE-BASED COMPARISON METRICS. METHODS WILL BE APPLIED TO SIMULATED AND PUBLIC METAGENOMIC DATA TO EVALUATE PERFORMANCE ON REAL DATA. TOP-PERFORMING METHODS WILL BE STRESS TESTED WITH AN EXPERIMENTALLY GENERATED BENCHMARKING DATASET DERIVED FROM CLOSELY RELATED SOIL SAMPLES ACROSS A FINE GRADIENT OF RESISTOME DIFFERENCE; THIS ANALYSIS WILL ALSO ALLOW ESTIMATION OF THE DISCRIMINATIVE LIMIT OF RESISTOME FINGERPRINTS. FINALLY, THE FINGERPRINTING METHODOLOGY AND BENCHMARKING DATASET WILL BE PACKAGED INTO AN ACCESSIBLE PIPELINE FOR FUTURE AND SUSTAINED USE.THIS PROJECT HAS DIRECT RELEVANCE TO THE GOALS OF PROGRAM AREA PRIORITY UNDERSTANDING ANTIMICROBIAL RESISTANCE, AS IT WILL PROVIDE A NOVEL METHOD TO TRACK AND MONITOR THE FATE, TRANSPORT, OR ROLE...OF ANTIMICROBIAL RESISTANCE...IN THE ENVIRONMENT.