pr2-Wormifier: A Bioinformatics Pipeline to Create Custom Reference Databases for Improved Metabarcoding of Marine...

Metabarcoding of environmental and ancient environmental DNA (eDNA and sedaDNA) is a powerful approach for studying and monitoring marine communities. However, its effectiveness is limited by the availability of comprehensive and well-curated reference databases, particularly for protists. Here, we introduce pr2-wormifier, a bioinformatics pipeline designed to create customized and improved reference databases for 18S rRNA-based metabarcoding. This pipeline integrates sequences from PR² and NCBI with taxonomic information from the World Register of Marine Species (WoRMS) and AlgaeBase, allowing for refined taxonomic assignments at the genus and species levels. pr2-wormifier enables users to tailor reference databases to specific taxonomic groups or geographic regions, enhancing the resolution and accuracy of biodiversity assessments. We benchmarked the pipeline using a sedimentary ancient DNA dataset from the Baltic Sea, focusing on marine protists, especially ciliates and dinoflagellates. The customized database generated by pr2-wormifier identified more sequences at the genus and species levels than PR² alone, while maintaining taxonomic consistency and quality. Our results demonstrate that pr2-wormifier addresses common limitations of existing databases, such as low taxonomic resolution and missing taxa and facilitates more reliable classification in metabarcoding studies. By enabling the creation of locally relevant, taxonomically curated databases, pr2-wormifier offers a flexible and scalable solution for improving the identification of protists in environmental and paleoenvironmental research.