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.Approach. 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.The initial optimization and standardization of the protocols described herein, and training of other NAHLN laboratories in these procedures will enable rapid implementation of NGS capability across NAHLN laboratories.