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Author Notes:

E-mail: Shanmuga.Sozhamannan@med.navy.mil

Conceived and designed the experiments: PEC LBP PSK AM TNB KABL TDR SS.

Performed the experiments: KMW AB SD MG SML CC LBP.

Analyzed the data: PEC AS CC AA KABL MEZ TDR SS.

Wrote the paper: PEC KABL MEZ TDR SS.

We would like to thank Tom Slezak and Marissa Chmiola for many useful comments on the manuscript and Brian Osborne and Christopher Dwan of the BioTeam for help with the bioinformatic pipeline used in this study.

The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.

The BDRD, NMRC authors are employees of the U.S. Government. This work was prepared as part of their official duties.

Title 17 U.S.C. §105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Research Funding:

This work was supported by the Transformational Medical Technologies Program under contracts TMTI0068_07_NM_T and TMTI-IB06RSQ002 through the Defense Threat Reduction Agency to TDR and SS respectively.

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • MULTIDISCIPLINARY SCIENCES
  • INHALATIONAL ANTHRAX
  • TECHNOLOGIES
  • CEREUS
  • BIOTERRORISM
  • EVOLUTION
  • STRAIN

Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

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Journal Title:

PLoS ONE

Volume:

Volume 5, Number 8

Publisher:

, Pages e12397-e12397

Type of Work:

Article | Final Publisher PDF

Abstract:

Background: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. Methodology/Principal Findings: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. Conclusions/Significance: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents.

Copyright information:

This is an Open Access work distributed under the terms of the Creative Commons Universal : Public Domain Dedication License (http://creativecommons.org/publicdomain/zero/1.0/).

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