Pyrosequencing is a cost-effective DNA sequencing technology that has many applications, including rapid genotyping of a broad spectrum of bacteria. When molecular typing requires to genotype multiple DNA stretches, several pyrosequencing primers could be used simultaneously but this would create overlapping primer-specific signals, which are visually uninterpretable. Accordingly, the objective was to develop a new method for signal processing (AdvISER-M-PYRO) to automatically analyze and interpret multiplex pyrosequencing signals. In parallel, the nucleotide dispensation order was improved by developing the SENATOR (SElecting the Nucleotide dispensATion Order) algorithm.