The Not-alike3 command pipeline finds genomic targets for the molecular diagnosis of diseases caused by pathogen microorganisms

DNA-based molecular techniques are crucial for the precise identification of microorganisms. Despite their speed and sensitivity, specificity hinges on primer or probe design. The search for unique sequences in organisms’ genomes has various applications such as microorganism identification. There are published bioinformatic tools that employ the concept of in silico genomic subtractive hybridization analysis to identify species-specific/unique regions within a genome of interest. However, they use deprecated tools and program[1]ming languages that are not currently used in bioinformatics, some of them are specialized or difficult to obtain because the repositories, where these tools were hosted, are not public or the web servers are currently down. To address these issues, we implemented the in silico genomic subtractive hybridization idea in a user-friendly, open-source, freely available, easy-to-install command pipeline application written in Python (called Not-alike3). We designed PCR primers over the sequence of unique regions identified in the genomes of two Mucorales species, Rhizopus oryzae and Cuninghamlla bertholletiae, employing Not-alike3 command pipeline; we made specificity tests to challenge these primers and we observed that they were species-specific.