Environmental sequencing has greatly expanded our knowledge of micro-eukaryotic diversity and ecology by revealing previously unknown lineages and their distribution. However, the value of these data is critically dependent on the quality of the reference databases used to assign an identity to environmental sequences. Existing databases contain errors and struggle to keep pace with rapidly changing eukaryotic taxonomy, the influx of novel diversity, and computational challenges related to assembling the high-quality alignments and trees needed for accurate characterization of lineage diversity. EukRef (eukref.org) is an ongoing community-driven initiative that addresses these challenges by bringing together taxonomists with expertise spanning the eukaryotic tree of life and microbial ecologists, who use environmental sequence data to develop reliable reference databases across the diversity of microbial eukaryotes. EukRef organizes and facilitates rigorous mining and annotation of sequence data by providing protocols, guidelines, and tools. The EukRef pipeline and tools allow users interested in a particular group of microbial eukaryotes to retrieve all sequences belonging to that group from International Nucleotide Sequence Database Collaboration (INSDC) (GenBank, the European Nucleotide Archive [ENA], or the DNA DataBank of Japan [DDBJ]), to place those sequences in a phylogenetic tree, and to curate taxonomic and environmental information for the group. We provide guidelines to facilitate the process and to standardize taxonomic annotations. The final outputs of this process are (1) a reference tree and alignment, (2) a reference sequence database, including taxonomic and environmental information, and (3) a list of putative chimeras and other artifactual sequences. These products will be useful for the broad community as they become publicly available (at eukref.org) and are shared with existing reference databases.

Department of Biological Sciences Mississippi State University Mississippi State Mississippi United States of America

Department of Biology and Centre for Comparative Genomics and Evolutionary Bioinformatics Dalhousie University Halifax Nova Scotia Canada

Department of Botany and Biodiversity Research Centre University of British Columbia Vancouver British Columbia Canada

Department of Marine Biology and Oceanography Institut de Ciències del Mar CSIC Barcelona Catalonia Spain

Department of Zoology University of British Columbia Vancouver British Columbia Canada

Departments of Marine Sciences and Ecology and Evolutionary Biology University of Connecticut Storrs United States of America

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Sorbonne Université CNRS Station Biologique de Roscoff UMR7144 Roscoff France

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