FOOD-BORNE PATHOGENS ENACT SUBSTANTIAL HARMS ON THE AMERICAN PEOPLE IN THE FORM OF ILLNESS, LOSTPRODUCTIVITY, AND EXPENSES RELATED TO MITIGATION AND REGULATORY COMPLIANCE. SURVEILLANCE ANDTRACING OF FOODBORNE PATHOGENS IS A KEY CONTROL STRATEGY, BUT ITS EFFICACY IS REDUCED BY THE LONGTIMESASSOCIATED WITH CURRENT CULTURE AND WHOLE-GENOME-SEQUENCING APPROACHES. RAPID, ACCURATEAND COMPREHENSIVE PATHOGEN DETECTION WOULD IMPROVE THE SAFETY AND LOWER THE COSTS OF OUR FOODSUPPLY.WE AIM TO DEVELOP A TARGETED METAGENOMICS METHODOLOGY THAT CAN RAPIDLY (&LT;24 HRS) AND PRECISELYIDENTIFY A BROAD RANGE OF FOODBORNE PATHOGENS FROM HETEROGENEOUS ENVIRONMENTAL SAMPLES. IN ORDERTO ACHIEVE THIS, WE PROPOSE TO COMBINE THE GENOMETRAKR AND NCBI REFSEQ DATABASES WITH CUTTINGEDGEBIOINFORMATICS TOOLS DEVELOPED BY THE PD THAT ACHIEVE SINGLE-NUCLEOTIDE RESOLUTION FROMAMPLICON SEQUENCING DATA OF FULL-LENGTH GENES TO IDENTIFY E. COLI AND SALMONELLA STRAINS TO THESEROVAR LEVEL (E.G. E. COLI O157:H7 OR S. ENTERICA HEIDELBERG). WE WILL VALIDATE THE RESOLUTION ANDACCURACY OF THIS NEW METHODOLOGY IN SILICO, ON ISOLATES OF VARIOUS PATHOGENIC SEROVARS, AND INENVIRONMENTAL SAMPLES OF VARIOUS TYPES FOR WHICH PATHOGEN PRESENCE AND IDENTITY WERE PREVIOUSLYESTABLISHED BY STANDARD CULTURE-BASED METHODS. OUR METHODOLOGY WILL BE DISTRIBUTED TO THE BROADERFOOD SAFETY COMMUNITY AS OPEN-SOURCE AND ACTIVELY-SUPPORTED SOFTWARE, ALONGSIDE EXTENSIVEDOCUMENTATION OF ITS EFFICACY AND BEST-PRACTICES GUIDANCE.SUCCESSFUL COMPLETION OF THIS PROJECT WILL YIELD A POWERFUL, USABLE, AND BROAD-SPECTRUM PATHOGENSURVEILLANCE TECHNIQUE THAT WILL IMPROVE FOOD SAFETY BY DETECTING FOODBORNE PATHOGENS BEFORE THEYREACH CONSUMERS, AND BY RAPIDLY TRACING OUTBREAKS TO THEIR SOURCE.