Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing

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.

Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness

“…Only 45 min of bioinformatics analysis (Pima version 01, https://github.com) using 120,000 basecalled nanopore reads was sufficient to assemble and confirm the absence of known AMR genes/markers associated with resistance to quinolones and tetracyclines ( Figure 1, panel B) (13). We detected no mutations in gyrA, gyrB, parC, or parE genes, identified no AMR genes contained in the Resfinder database (https://cge.…”

“…Although genetic analysis is useful for detection of known AMR genes/markers in the B. anthracis genome, phenotypic susceptibility testing by BMD remains essential to detect functional resistance (7,13). By using the conventional BMD method, we found that strain Ba0914 was susceptible to ciprofloxacin, levofloxacin, tetracycline, doxycycline, penicillin, and amoxicillin.…”

“…All laboratory work with the B. anthracis isolate and nanopore sequencing was performed inside a class II type A2 biological safety cabinet located in a US Federal Select Agent Program registered Biosafety Level 3 laboratory. We performed rapid nanopore sequencing as described by Gargis et al (13), including silica membrane-based genomic DNA (gDNA) extraction, except that a bead-beating step was added to speed cell lysis. We extracted Ba0914 gDNA from colonies of an overnight agar culture in 75 min.…”

“…The technology enables realtime sequencing, including direct pathogen identification from patient specimens, and holds the promise for future point-of-care applications that speed laboratory results reporting (11,12). Portable WGS instruments are advantageous for laboratories with limited space and remove the need to transfer DNA out of high-containment laboratories for sequencing, mitigating exposure risks to personnel (13).…”

“…CDC described a rapid nanopore sequencing approach and custom bioinformatics pipeline for B. anthracis that yielded complete chromosome and plasmid assemblies, and detected known AMR genes and mutations in avirulent laboratory strains (13). On the morning of August 2, 2019, our laboratory received a B. anthracis culture isolate (Ba0914) from a naturally occurring human anthrax case in Texas.…”