Vázquez González, LaraRegueira Iglesias, AlbaBalsa Castro, CarlosVila Blanco, NicolásTomás Carmona, InmaculadaCarreira Nouche, María José2025-03-312025-03-312024-05-14Vázquez-González, L., Regueira-Iglesias, A., Balsa-Castro, C. et al. PrimerEvalPy: a tool for in-silico evaluation of primers for targeting the microbiome. BMC Bioinformatics 25, 189 (2024). https://doi.org/10.1186/s12859-024-05805-7https://hdl.handle.net/10347/40636Background: The selection of primer pairs in sequencing‑based research can greatly influence the results, highlighting the need for a tool capable of analysing their performance in-silico prior to the sequencing process. We therefore propose PrimerEvalPy, a Python‑based package designed to test the performance of any primer or primer pair against any sequencing database. The package calculates a coverage metric and returns the amplicon sequences found, along with information such as their average start and end positions. It also allows the analysis of coverage for different taxonomic levels. Results: As a case study, PrimerEvalPy was used to test the most commonly used primers in the literature against two oral 16S rRNA gene databases containing bacteria and archaea. The results showed that the most commonly used primer pairs in the oral cavity did not match those with the highest coverage. The best performing primer pairs were found for the detection of oral bacteria and archaea. Conclusions: This demonstrates the importance of a coverage analysis tool such as PrimerEvalPy to find the best primer pairs for specific niches. The software is available under the MIT licence at https://gitlab.citius.usc.es/lara.vazquez/PrimerEvalPy.eng© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holderAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/BioinformaticsPrimer165 rRNA geneMicrobiomeSequence analysisjournal article10.1186/s12859-024-05805-71471-2105open access