ABSTRACTPlasmids are extrachromosomal genetic elements replicating independently of the chromosome which play a vital role in the environmental adaptation of bacteria. Due to potential mobilization or conjugation capabilities, plasmids are important genetic vehicles for antimicrobial resistance genes and virulence factors with huge and increasing clinical implications. They are therefore subject to large genomic studies within the scientific community worldwide. As a result of rapidly improving next generation sequencing methods, the amount of sequenced bacterial genomes is constantly increasing, in turn raising the need for specialized tools to (i) extract plasmid sequences from draft assemblies, (ii) derive their origin and distribution, and (iii) further investigate their genetic repertoire. Recently, several bioinformatic methods and tools have emerged to tackle this issue; however, a combination of both high sensitivity and specificity in plasmid sequence identification is rarely achieved in a taxon-independent manner. In addition, many software tools are not appropriate for large high-throughput analyses or cannot be included into existing software pipelines due to their technical design or software implementation. In this study, we investigated differences in the replicon distributions of protein-coding genes on a large scale as a new approach to distinguish plasmid-borne from chromosome-borne contigs. We defined and computed statistical discrimination thresholds for a new metric: the replicon distribution score (RDS) which achieved an accuracy of 96.6%. The final performance was further improved by the combination of the RDS metric with heuristics exploiting several plasmid specific higher-level contig characterizations. We implemented this workflow in a new high-throughput taxon-independent bioinformatics software tool called Platon for the recruitment and characterization of plasmid-borne contigs from short-read draft assemblies. Compared to PlasFlow, Platon achieved a higher accuracy (97.5%) and more balanced predictions (F1=82.6%) tested on a broad range of bacterial taxa and better or equal performance against the targeted tools PlasmidFinder and PlaScope on sequenced E. coli isolates. Platon is available at: platon.computational.bioData SummaryPlaton was developed as a Python 3 command line application for Linux.The complete source code and documentation is available on GitHub under a GPL3 license: https://github.com/oschwengers/platon and platon.computational.bio.All database versions are hosted at Zenodo: DOI 10.5281/zenodo.3349651.Platon is available via bioconda package platonPlaton is available via PyPI package cb-platonBacterial representative sequences for UniProt’s UniRef90 protein clusters, complete bacterial genome sequences from the NCBI RefSeq database, complete plasmid sequences from the NCBI genomes plasmid section, created artificial contigs, RDS threshold metrics and raw protein replicon hit counts used to create and evaluate the marker protein sequence database are hosted at Zenodo: DOI 10.5281/zenodo.375916924 Escherichia coli isolates sequenced with short read (Illumina MiSeq) and long read sequencing technologies (Oxford Nanopore Technology GridION platform) used for real data benchmarks are available under the following NCBI BioProjects: PRJNA505407, PRJNA387731Impact StatementPlasmids play a vital role in the spread of antibiotic resistance and pathogenicity genes. The increasing numbers of clinical outbreaks involving resistant pathogens worldwide pushed the scientific community to increase their efforts to comprehensively investigate bacterial genomes. Due to the maturation of next-generation sequencing technologies, nowadays entire bacterial genomes including plasmids are sequenced in huge scale. To analyze draft assemblies, a mandatory first step is to separate plasmid from chromosome contigs. Recently, many bioinformatic tools have emerged to tackle this issue. Unfortunately, several tools are implemented only as interactive or web-based tools disabling them for necessary high-throughput analysis of large data sets. Other tools providing such a high-throughput implementation however often come with certain drawbacks, e.g. providing taxon-specific databases only, not providing actionable, i.e. true binary classification or achieving biased classification performances towards either sensitivity or specificity.Here, we introduce the tool Platon implementing a new replicon distribution-based approach combined with higher-level contig characterizations to address the aforementioned issues. In addition to the plasmid detection within draft assemblies, Platon provides the user with valuable information on certain higher-level contig characterizations. We show that Platon provides a balanced classification performance as well as a scalable implementation for high-throughput analyses. We therefore consider Platon to be a powerful, species-independent and flexible tool to scan large amounts of bacterial whole-genome sequencing data for their plasmid content.
Identification of Novel Conotoxin Precursors from the Cone Snail Conus spurius by High-Throughput RNA Sequencing