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bioRxiv · 10.1101/2025.07.08.663663

MicrobiomePrime: A primer pair selection tool for microbial source tracking validated on a comprehensive collection of animal gut and fecal waste microbiomes

Zlender, T.·Brezocnik, L.·Podgorelec, V.·Rupnik, M.
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Abstract

Identifying primer pairs for use in microbial source tracking (MST) can be challenging. The primer pairs must amplify markers with high sensitivity and specificity for a particular microbiota source, while factors such as melting temperature, secondary structure formation, and amplicon length must be carefully considered to ensure efficient amplification. To accomplish this, previous MST studies relied on complex, multi-step analysis, which was labor-intensive and time-consuming. In this paper, we present MicrobiomePrime, a tool for identifying primer pairs for amplification of source-associated markers by analyzing amplicon sequences. Our approach is based on splitting amplicon sequences from target sources into K-mers, which are treated as potential primers in an in silico PCR. As proof of concept, we applied the tool to identify markers associated with pig feces, generating thousands of primer pairs. Among nine primer pairs selected for laboratory validation, six primer pairs exhibited in vitro specificity higher than 93 %. Among them, four were 100 % specific. While originally developed for MST, MicrobiomePrime has broader applicability, enabling the design of primers targeting any molecular marker based on amplicon sequencing data. MicrobiomePrime is available on GitHub: https://github.com/tanjazlender/MicrobiomePrime. Author summaryDetecting molecular markers is one of the key approaches for identifying specific traits or differences. One approach for the detection of molecular markers involves amplifying them with primer pairs that are highly specific, sensitive and carefully designed to ensure optimal assay performance. Existing methods for designing these primers are often labor-intensive, involving complex, multi-step analyses. To address this challenge, we developed MicrobiomePrime, a tool that offers a streamlined design of source-associated primer pairs by analyzing sequencing data. It eliminates the need for complex multi-step analysis and enables a rapid development of novel PCR assays for the detection of molecular markers. The method is broadly applicable, however, we have developed it for microbial source tracking sources of fecal contamination. Identifying sources of contamination is namely essential for controlling microbial spread, improving sanitation practices, and ensuring effective remediation of affected environments.

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Zlender, T., Brezocnik, L., Podgorelec, V., Rupnik, M.. 2025-07-11. MicrobiomePrime: A primer pair selection tool for microbial source tracking validated on a comprehensive collection of animal gut and fecal waste microbiomes. https://doi.org/10.1101/2025.07.08.663663

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