Comparison of Bioinformatic Pipelines for eDNA Metabarcoding Data Analysis of Fish Populations

Environmental DNA (eDNA) metabarcoding has gained popularity as a biomonitoring tool, leading to the emergence of various bioinformatic pipelines. However, comparisons are essential to assessing the reliability and similarity of results. In this study, we compared five bioinformatic pipelines (Anacapa, Barque, metaBEAT, MiFish, and SEQme) using samples collected from three reservoirs in the Czech Republic during the autumn and summer seasons. Negative and positive controls were used to monitor potential contamination during sample processing. eDNA was amplified, targeting the 12S fish rRNA gene, sequenced, and analyzed with the selected bioinformatic pipelines. Statistical analyses were applied to assess result similarity, including the number of detected taxa, read count, alpha and beta diversities, and the Mantel test. Our findings showed consistent taxa detection across pipelines, with increased sensitivity compared to traditional methods. Alpha and beta diversities and the Mantel test also exhibited significant similarities between pipelines. Divergences were observed based on the reservoir, season, and their interaction. In conclusion, the choice of bioinformatic pipeline did not significantly affect metabarcoding outcomes or their ecological interpretation.