Converting a pyrosequencing signal into a nucleotide sequence appears highly challenging when signal intensities are low (unitary peak heights ) or when complex signals are produced by several target amplicons. In these cases, the pyrosequencing software fails to provide correct nucleotide sequences. Accordingly, the objective was to develop the AdvISER-PYRO algorithm, performing an automated, fast and reliable analysis of pyrosequencing signals that circumvents those limitations.