Plasmid detection and assembly in genomic and metagenomic data sets

Abstract

Although plasmids are important for bacterial survival and adaptation, plasmid detection and assembly from genomic, let alone metagenomic, samples remain challenging. The recently developed plasmidSPAdes assembler addressed some of these challenges in the case of isolate genomes but stopped short of detecting plasmids in metagenomic assemblies, an untapped source of yet to be discovered plasmids. We present the metaplasmidSPAdes tool for plasmid assembly in metagenomic data sets that reduced the false positive rate of plasmid detection compared with the state-of-the-art approaches. We assembled plasmids in diverse data sets and have shown that thousands of plasmids remained below the radar in already completed genomic and metagenomic studies. Our analysis revealed the extreme variability of plasmids and has led to the discovery of many novel plasmids (including many plasmids carrying antibiotic-resistance genes) without significant similarities to currently known ones.

Figure 1.

Iterative plasmid detection in the assembly graph. (A) The assembly graph Graph with three dotted edges representing edges with the lowest coverage. (B) Removal of three edges with the lowest coverage from Graph reveals a plasmid (cyclocontig) shown in blue. The three edges on the graph in A now represent a single dashed edge that has the lowest coverage in Graph. (C) The same graph after the second iteration of metaplasmidSPAdes that removes the dashed edge with the lowest coverage and reveals a plasmid (connected component) shown in red.

Figure 2.

The scatter plot of the span and identity for all 2280 unique cyclocontigs in the ISOLATES data set reconstructed by plasmidSPAdes⁺ (A) and the Venn diagram for cyclocontigs identified as plasmids by plasmidVerify, cyclocontigs identified as plasmids by BLAST (span >10%), and cyclocontigs containing ARGs (B). (A) Each dot represents a cyclocontig reported by plasmidSPAdes and verified by plasmidVerify. Red dots represent cyclocontigs containing antibiotic-resistance genes (ARGs). Green dots represent cyclocontigs classified as viral sequences. (B) The Venn diagram illustrates that the HMM-based approach in metaplasmidSPAdes identifies many plasmids with important phenotypes that are missed by a straightforward BLAST-based approach.