Published by Cold Spring Harbor Laboratory Press. Bacterial isolates can contain any number of plasmids and assembly remains complicated due to the presence of repetitive elements. However, analysing plasmid content remains difficult due to incomplete assembly of plasmids. 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. Large-scale bacterial population genetics studies are now routine due to cost-effective Illumina short-read sequencing. 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. Samples were processed in 10 plates of 96 plasmids each with the plexWell 384 Library Preparation Kit. This plasmid is available through Addgene. Read distributions by plate and well position for 960 plasmids sequencing in a single Illumina MiSeq (2 x 250 bp) run. Tyler Jacks's lab contains the insert Puromycin Resistance and is published in Sci Rep. 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. Scalable, true multiplexed library preparation enables high-performance, high-throughput plasmid sequencing. 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. Although plasmids are important for bacterial survival and adaptation, plasmid detection and assembly from genomic, let alone metagenomic, samples remain challenging.
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