Nation-scale recovery of complete environmental plasmids pushes the boundaries of plasmid diversity through long-read sequencing

Plasmids are important agents of prokaryotic evolution and yet existing reference databases remain biased towards cultivable species, limiting our knowledge of their function and ecology in the environment. Here we present a major expansion of known plasmid diversity, through curation and annotation of 8,744 topologically complete plasmids from 9,972 short- and 154 long-read metagenomes as part of the Microflora Danica project. These were found to increase plasmid taxonomic unit diversity by 15% and 99% relative to all and environmental plasmids, respectively, from public sources. In particular, we identify a novel lineage of plasmids in the genus Methanosarcina, encoding genes associated with methanogenesis and cell surface properties, expanding our understanding of chromosome-plasmid complementarity and its potential contribution to host biology. Combining our plasmids with public sources, we curated a set of 17 replication-associated proteins that could be found in 82% of known plasmids and can serve as hallmarks for future detection of plasmids. We present this resource, and a tool for confirming topological completeness of plasmid assemblies, as a toolkit for future plasmid discovery.