Protists are unicellular eukaryotes that exhibit different modes of nutrition, they can be Phototrophic (algae), Heterotrophic, or even Mixotrophic. Thus, they are important contributors to the aquatic food web, and the main grazers of bacteria in aquatic environments. Although some protists can be identified using microscopy techniques, Heterotrophic nanoflagellates (HNF) are morphologically similar despite the immense phylogenetic diversity. For this reason, it is challenging to isolate and culture them based on their morphology. Metagenomics offers a culture-free alternative towards the recovery of genomes from the vast amounts of metagenomic sequence data that is becoming available and has been transformative for understanding prokaryotic diversity.

Efforts to recover eukaryotic protist genomes, which may be hundreds of times larger than prokaryotic genomes, have remained limited due absence of long time series datasets with sufficient sequencing depth. In effect, genomes of abundant protists in freshwater habitats remain largely unknown. Here we assembled and binned 1096 metagenomes from decade-long time series from four lakes (Stechlin, Grosse Fuchskuhle, Breiter Luzin, and Tiefwaren) and assessed the recovery of the largest set of eukaryotic metagenome-assembled genomes (MAGs) from freshwater lakes. Close examination of over 1000 putative eukaryotic MAGs indicated several types of contaminations, from prokaryotes, viruses, and even multiple, taxonomically distinct eukaryotes within these metagenomic bins. Using conservative filtration thresholds and manual curation, we recovered the largest number of protist genomes from freshwater lakes, namely, 280 taxonomically coherent, high-quality eukaryotic MAGs that ranged from 5-50 MB in size.