MultiBarcodeTools: Easy selection of optimal primers for eDNA multi‐metabarcoding

Abstract

AbstractMetabarcoding of environmental DNA (eDNA) is a revolutionary technique for comprehensive and efficient monitoring of ecosystems, including invasive and endangered species. Although numerous universal primers have been developed for eDNA metabarcoding, no single primer can amplify and discriminate all species in any environment. Parallel metabarcoding using multiple primer sets, or multi‐metabarcoding, is expected to overcome this limitation; however, it is not easy to select optimal primers from dozens of existing primers considering various research purposes and sequence platform availabilities. Herein, we developed MultiBarcodeSelector, which automatically recommends the optimal combination of fish eDNA multi‐metabarcoding primers, and MultiBarcodeMap, which pre‐computes region‐specific optimal primers. Furthermore, for users who want to target non‐fish species, use original primers, and develop their own databases, we also provide a stand‐alone MultiBarcodePipeline. We show that multi‐metabarcoding substantially enhances the performance of eDNA community analysis. In our assessment that targeted fish species in various aquatic regions of Japan, an extra barcode suggested by the MultiBarcodeTools significantly reduced the proportion of undistinguishable species. Our MultiBarcodeTools will benefit various biodiversity research studies, including species detection, monitoring, conservation, and economic utilization. MultiBarcodeSelector and MultiBarcodeMap are available at https://multibarcode.k.u‐tokyo.ac.jp, and MultiBarcodePipeline is available at https://doi.org/10.5281/zenodo.10007568.