Agricultural high throughput diagnostics need to be fast, accurate and have multiplexing capacity. Metagenomic sequencing is being widely evaluated for plant and animal diagnostics. Bioinformatic analysis of metagenomic sequence data has been a bottleneck for diagnostic analysis due to the size of the data files. Most available tools for analyzing high-throughput sequencing (HTS) data require that the user have computer coding skills and access to high-performance computing. To overcome constraints to most sequencing-based diagnostic pipelines today, we have developed Microbe Finder (MiFi®). MiFi® is a web application for quick detection and identification of known pathogen species/strains in raw, unassembled HTS metagenomic data. HTS-based diagnostic tools developed through MiFi® must pass rigorous validation, which is outlined in this manuscript. MiFi® allows researchers to collaborate in the development and validation of HTS-based diagnostic assays using MiProbe™, a platform used for developing pathogen-specific e-probes. Validated e-probes are made available to diagnosticians through MiDetect™. Here we describe the e-probe development, curation and validation process of MiFi® using grapevine pathogens as a model system. MiFi® can be used with any pathosystem and HTS platform after e-probes have been validated.
Keywords: Microbe Finder (MiFi); assay validation; diagnostics; metagenomics; nucleic acid; pathogens; sensitivity and specificity; sequencing; taxonomy.