Fig. 1

Outline of the workflow of AmpliDiff. A Pre-processing consists of building a reference set from a database of genomes (e.g. GISAID) and applying multiple sequence alignment to obtain multiple aligned genomes. B AmpliDiff builds a database of feasible primers that can be considered during the greedy amplicon selection (iii) step. C Feasible amplicon candidates are extracted from the multiple aligned genomes, and for every amplicon, the pairs of genomes it can differentiate are stored. D Greedy amplicon and primer selection until either all pairs of genomes can be differentiated, a user-defined number of amplicons is found, or no more feasible amplicons exist. AmpliDiff outputs the selected amplicons and corresponding primer sets