plsMD: A plasmid reconstruction tool from short-read assemblies

While whole genome sequencing (WGS) has become a cornerstone of antimicrobial resistance (AMR) surveillance, the reconstruction of plasmid sequences from short-read WGS data remains a challenge due to repetitive sequences and assembly fragmentation. Current computational tools for plasmid identification and binning, such as PlasmidFinder, cBAR, PlasmidSPAdes, and Mob-recon, have limitations in reconstructing full plasmid sequences, hindering downstream analyses like phylogenetic studies and AMR gene tracking. To address this gap, we present plsMD, a novel tool designed for full plasmid reconstruction from short-read assemblies. plsMD integrates Unicycler assemblies with replicon and full plasmid sequence databases (PlasmidFinder and PLSDB) to guide plasmid reconstruction through a series of contig manipulations. Using a dataset of 83 samples containing 223 plasmids, plsMD outperformed existing tools, achieving excellent recall and precision of 91.2% and 93.6%, respectively, for plasmid reconstruction, and high sensitivity and specificity of 94.5% and 99.5% for plasmid-chromosomal contig separation. plsMD supports two usage modalities: single-sample analysis for plasmid reconstruction and gene annotation, and multi-sample analysis for phylogenetic investigations of plasmid transmission. This computational tool represents a significant advancement in plasmid analysis, offering a robust solution for utilizing existing short-read WGS data to study plasmid-mediated AMR spread and evolution.