Abstract Chapter “DNA metabarcode choice and design” develops the properties of the ideal metabarcode in a given context, including conservation of the primer annealing regions and resolution power across the target taxonomic group of interest. It also highlights the experimental constraints influencing the choice of a metabarcode in practice. A detailed tutorial illustrates how to design and test metabarcoding primers in silico with the programs ecoPrimers, ecoPCR, and the software suite OBITools. Command lines and example files are provided to design and test universal metabarcoding primers for Bacteria. Chapter 2 also gives statistics about the taxonomic resolution and primer conservation of more than 60 metabarcodes available for DNA metabarcoding analysis of a wide range of taxonomic groups.