**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** TRANSBOUNDARY AND EMERGING INFECTIOUS DISEASES POSE A CONSTANT THREAT TO THE U.S. LIVESTOCK INDUSTRY. RAPID METHODS FOR PATHOGEN DETECTION SUCH AS REAL-TIME PCR ARE WIDELY UTILIZED IN DIAGNOSTIC LABORATORIES IN THE U.S. HOWEVER, AS THESE ASSAYS ARE HIGHLY SPECIFIC TO PREVIOUSLY KNOWN AND CHARACTERIZED PATHOGENS, THEY MAY FAIL TO DETECT NEWLY EMERGING PATHOGENS OR VARIANTS OF KNOWN PATHOGENS THAT ARE CONSTANTLY EMERGING THROUGHOUT THE WORLD. THESE CHALLENGES COULD BE OVERCOME WITH THE USE OF NEXT GENERATION SEQUENCING (NGS) TECHNOLOGIES, WHICH PROVIDE A MEANS FOR RAPID AND SIMULTANEOUS DETECTION, IDENTIFICATION AND GENETIC CHARACTERIZATION OF PATHOGENS. THE MAJOR BOTTLENECKS THAT CURRENTLY HINDER THE WIDE USE OF NGS IN ROUTINE PATHOGEN DIAGNOSTICS ARE: I. THE LACK OF STANDARDIZED NGS PROTOCOLS AND PROCEDURES THAT CAN BE USED ACROSS DIFFERENT LABORATORIES; II. THE NEED FOR HIGHLY SPECIALIZED BIOINFORMATICS CAPABILITIES FOR NGS DATA ANALYSIS, INTERPRETATION, AND REPORTING; AND III. THE LACK OF TRAINING IN NGS PROCEDURES AND BIOINFORMATICS ACROSS DIFFERENT DIAGNOSTIC LABORATORIESOVERALL GOAL. THE OVERARCHING GOAL OF THIS INTEGRATED PROJECT IS TO ENHANCE THE NATIONAL ANIMAL HEALTH LABORATORY NETWORK (NAHLN) DIAGNOSTIC CAPABILITIES AND EMERGING DISEASE PREPAREDNESS THROUGH THE DEVELOPMENT OF NEXT-GENERATION SEQUENCING (NGS) ASSAYS AND BIOINFORMATICS ANALYSIS PIPELINES FOR THE DETECTION AND CHARACTERIZATION OF TRANSBOUNDARY AND EMERGING PATHOGENS OF CATTLE.SPECIFIC OBJECTIVES. THE OBJECTIVES OF THE PROPOSED STUDY ARE: (1) TO DEVELOP AND OPTIMIZE NGS-BASED DIAGNOSTIC ASSAYS THAT WILL ALLOW EARLY DETECTION OF TRANSBOUNDARY AND/OR EMERGING DISEASES OF CATTLE; (2) TO DEVELOP AND OPTIMIZE STREAMLINED BIOINFORMATICS PIPELINES FOR TARGETED AND RANDOM NGS DIAGNOSTIC APPLICATIONS FOR CATTLE; AND (3) TO ESTABLISH NGS-BASED DIAGNOSTIC CAPABILITY THROUGHOUT THE NATIONAL ANIMAL HEALTH LABORATORY NETWORK.IN OBJECTIVE 1 (O1), WE WILL DEVELOP AND OPTIMIZE TARGETED (AMPLICON-BASED) AND RANDOM (METAGENOMICS-BASED) ASSAYS AND PROCEDURES FOR USE IN THE DIAGNOSIS OF VESICULAR (I.E FMDV), RESPIRATORY (BOVINE RESPIRATORY DISEASE COMPLEX [BRD] PATHOGENS) AND/OR ENTERIC DISEASES OF CATTLE. IN O2, WE WILL DEVELOP BIOINFORMATICS ANALYSIS PIPELINES FOR RAPID DATA ANALYSIS AND RESULT REPORTING FOR THE ASSAYS DEVELOPED IN O1. ADDITIONALLY, A RAPID REFERENCE-BASED ASSEMBLY PIPELINE FOR THE TARGETED SEQUENCING APPROACHES WILL BE DEVELOPED AND OPTIMIZED. IN O3, WE WILL TRAIN NAHLN LABORATORIES IN NGS PROCEDURES AND BIOINFORMATICS ANALYSIS IN ORDER TO ESTABLISH AND IMPLEMENT THE NGS DIAGNOSTIC TOOLS DEVELOPED IN OBJECTIVES 1 AND 2 ACROSS THE NAHLN.EARLY AND RAPID DETECTION OF TRANSBOUNDARY AND EMERGING INFECTIOUS DISEASES ARE AT THE FOREFRONT OF SUCCESSFUL DISEASE CONTROL AND PREVENTION STRATEGIES. THE PROPOSED PROJECT WILL DIRECTLY ENHANCE THIS CAPACITY BY STANDARDIZING AND OPTIMIZING NGS PROTOCOLS AND PROCEDURES FOR BROAD PATHOGEN DETECTION IN CATTLE.