Abstract
Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5 h) and B. anthracis (8.5 h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency.
Highlights
Www.nature.com/scientificreports health concern[12,13]
We developed a custom bioinformatics pipeline to compare the genome assemblies from standardized replicate nanopore sequencing experiments to define sequencing quality metrics, establish performance characteristics, and demonstrate the feasibility of rapid long-read Whole genome sequencing (WGS) for Y. pestis and B. anthracis
Detection of genetic manipulation and/or unexpected antimicrobial resistance (AMR) profiles could signal the introduction of plasmids, AMR genes, or other evidence of engineering
Summary
Www.nature.com/scientificreports health concern[12,13]. Y. pestis is intrinsically susceptible to every antibiotic recommended for human plague therapy, but a few Y. pestis isolates with transferable plasmid-mediated resistance were reported from Madagascar[13,14,15,16]. Many AMR gene regions are flanked by repetitive insertion sequences (IS), and short-read sequencing cannot span these regions[25] This can result in inaccurate chromosome or plasmid assemblies, including multiple copies of a repeat region collapsed into one location[25,27]. We developed a custom bioinformatics pipeline to compare the genome assemblies from standardized replicate nanopore sequencing experiments to define sequencing quality metrics, establish performance characteristics, and demonstrate the feasibility of rapid long-read WGS for Y. pestis and B. anthracis. The resulting same-day laboratory workflow for DNA isolation, library preparation, nanopore sequencing, and bioinformatics analysis is timely and effective for the rapid detection of known markers related to AMR and evidence of genetic engineering in bacterial biothreat pathogens
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