Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing
Abstract
Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray inmore »
- Authors:
-
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- University of Tennessee, Knoxville, TN (United States)
- Publication Date:
- Research Org.:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1627636
- Grant/Contract Number:
- AC52-07NA27344; AC05-00OR22725; HSHQDC-09-X-00168
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Volume: 8; Journal Issue: 9; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; bacillus anthracis; genomics; aerosols; bacillus cereus; bacillus thuringiensis; bacterial genomics; sequence alignment; census
Citation Formats
Be, Nicholas A., Thissen, James B., Gardner, Shea N., McLoughlin, Kevin S., Fofanov, Viacheslav Y., Koshinsky, Heather, Ellingson, Sally R., Brettin, Thomas S., Jackson, Paul J., and Jaing, Crystal J. Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing. United States: N. p., 2013.
Web. doi:10.1371/journal.pone.0073455.
Be, Nicholas A., Thissen, James B., Gardner, Shea N., McLoughlin, Kevin S., Fofanov, Viacheslav Y., Koshinsky, Heather, Ellingson, Sally R., Brettin, Thomas S., Jackson, Paul J., & Jaing, Crystal J. Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing. United States. https://doi.org/10.1371/journal.pone.0073455
Be, Nicholas A., Thissen, James B., Gardner, Shea N., McLoughlin, Kevin S., Fofanov, Viacheslav Y., Koshinsky, Heather, Ellingson, Sally R., Brettin, Thomas S., Jackson, Paul J., and Jaing, Crystal J. Mon .
"Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing". United States. https://doi.org/10.1371/journal.pone.0073455. https://www.osti.gov/servlets/purl/1627636.
@article{osti_1627636,
title = {Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing},
author = {Be, Nicholas A. and Thissen, James B. and Gardner, Shea N. and McLoughlin, Kevin S. and Fofanov, Viacheslav Y. and Koshinsky, Heather and Ellingson, Sally R. and Brettin, Thomas S. and Jackson, Paul J. and Jaing, Crystal J.},
abstractNote = {Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.},
doi = {10.1371/journal.pone.0073455},
journal = {PLoS ONE},
number = 9,
volume = 8,
place = {United States},
year = {Mon Sep 09 00:00:00 EDT 2013},
month = {Mon Sep 09 00:00:00 EDT 2013}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
What Sets Bacillus anthracis Apart from Other Bacillus Species?
journal, October 2009
- Kolstø, Anne-Brit; Tourasse, Nicolas J.; Økstad, Ole Andreas
- Annual Review of Microbiology, Vol. 63, Issue 1
Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development
journal, January 2012
- Han, Tao; Chang, Ching-Wei; Kwekel, Joshua C.
- BMC Genomics, Vol. 13, Issue 1
Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation
journal, March 2011
- Rasko, D. A.; Worsham, P. L.; Abshire, T. G.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 12
Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen
journal, October 2002
- Quinn, Conrad P.; Semenova, Vera A.; Elie, Cheryl M.
- Emerging Infectious Diseases, Vol. 8, Issue 10
Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness
journal, November 2012
- Buzard, Gregory S.; Baker, Daniel; Wolcott, Mark J.
- Forensic Science International, Vol. 223, Issue 1-3
A microbial detection array (MDA) for viral and bacterial detection
journal, January 2010
- Gardner, Shea N.; Jaing, Crystal J.; McLoughlin, Kevin S.
- BMC Genomics, Vol. 11, Issue 1
Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing
journal, August 2010
- Chen, Peter E.; Willner, Kristin M.; Butani, Amy
- PLoS ONE, Vol. 5, Issue 8
Bacillus anthracis: toxicology, epidemiology and current rapid-detection methods
journal, November 2005
- Edwards, Katie A.; Clancy, Harriet A.; Baeumner, Antje J.
- Analytical and Bioanalytical Chemistry, Vol. 384, Issue 1
Implications of Limits of Detection of Various Methods for Bacillus anthracis in Computing Risks to Human Health
journal, July 2009
- Herzog, Amanda B.; McLennan, S. Devin; Pandey, Alok K.
- Applied and Environmental Microbiology, Vol. 75, Issue 19
Anthrax Toxin: Receptor Binding, Internalization, Pore Formation, and Translocation
journal, June 2007
- Young, John A. T.; Collier, R. John
- Annual Review of Biochemistry, Vol. 76, Issue 1
Rapidly Progressive, Fatal, Inhalation Anthrax-like Infection in a Human: Case Report, Pathogen Genome Sequencing, Pathology, and Coordinated Response
journal, November 2011
- Wright, Angela M.; Beres, Stephen B.; Consamus, Erin N.
- Archives of Pathology & Laboratory Medicine, Vol. 135, Issue 11
Characterization of Bacillus anthracis-Like Bacteria Isolated from Wild Great Apes from Cote d'Ivoire and Cameroon
journal, July 2006
- Klee, S. R.; Ozel, M.; Appel, B.
- Journal of Bacteriology, Vol. 188, Issue 15
Detection technologies for Bacillus anthracis: Prospects and challenges
journal, July 2010
- Rao, Shilpakala Sainath; Mohan, Ketha V. K.; Atreya, Chintamani D.
- Journal of Microbiological Methods, Vol. 82, Issue 1
A Functional Gene Array for Detection of Bacterial Virulence Elements
journal, May 2008
- Jaing, Crystal; Gardner, Shea; McLoughlin, Kevin
- PLoS ONE, Vol. 3, Issue 5
Detection of Virulence-Associated Genes in Clinical Isolates of Bacillus anthracis by Multiplex PCR and DNA Probes
journal, January 2009
- Kumar, Sanjay
- Journal of Microbiology and Biotechnology, Vol. 19
Rapid detection methods for Bacillus anthracis in environmental samples: a review
journal, January 2012
- Irenge, Léonid M.; Gala, Jean-Luc
- Applied Microbiology and Biotechnology, Vol. 93, Issue 4
Investigation of Bioterrorism-Related Anthrax, United States, 2001: Epidemiologic Findings
journal, October 2002
- Jernigan, Daniel B.; Raghunathan, Pratima L.; Bell, Beth P.
- Emerging Infectious Diseases, Vol. 8, Issue 10
DINAMelt web server for nucleic acid melting prediction
journal, July 2005
- Markham, N. R.; Zuker, M.
- Nucleic Acids Research, Vol. 33, Issue Web Server
The Bacillus anthracis spore
journal, December 2009
- Driks, Adam
- Molecular Aspects of Medicine, Vol. 30, Issue 6
Efficient implementation of lazy suffix trees
journal, January 2003
- Giegerich, R.; Kurtz, S.; Stoye, J.
- Software: Practice and Experience, Vol. 33, Issue 11
Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases
journal, September 2004
- Keys, Carrina J.; Dare, Diane J.; Sutton, Helen
- Infection, Genetics and Evolution, Vol. 4, Issue 3
Experimental and analytical tools for studying the human microbiome
journal, December 2011
- Kuczynski, Justin; Lauber, Christian L.; Walters, William A.
- Nature Reviews Genetics, Vol. 13, Issue 1
Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection
journal, May 2008
- Sohni, Youvraj; Kanjilal, Sagarika; Kapur, Vivek
- Clinical Biochemistry, Vol. 41, Issue 7-8
Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing
journal, January 2010
- Cummings, Craig A.; Bormann-Chung, Christina A.; Fang, Rixun
- Investigative Genetics, Vol. 1, Issue 1
The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria
journal, May 2003
- Read, Timothy D.; Peterson, Scott N.; Tourasse, Nicolas
- Nature, Vol. 423, Issue 6935
Efficient Implementation of Lazy Suffix Trees
book, January 1999
- Giegerich, Robert; Kurtz, Stefan; Stoye, Jens
- Algorithm Engineering
Rapid detection methods for Bacillus anthracis in environmental samples: a review
journal, January 2012
- Irenge, Léonid M.; Gala, Jean-Luc
- Applied Microbiology and Biotechnology, Vol. 93, Issue 4
Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection
journal, May 2008
- Sohni, Youvraj; Kanjilal, Sagarika; Kapur, Vivek
- Clinical Biochemistry, Vol. 41, Issue 7-8
Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness
journal, November 2012
- Buzard, Gregory S.; Baker, Daniel; Wolcott, Mark J.
- Forensic Science International, Vol. 223, Issue 1-3
The Bacillus anthracis spore
journal, December 2009
- Driks, Adam
- Molecular Aspects of Medicine, Vol. 30, Issue 6
Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases
journal, September 2004
- Keys, Carrina J.; Dare, Diane J.; Sutton, Helen
- Infection, Genetics and Evolution, Vol. 4, Issue 3
Detection technologies for Bacillus anthracis: Prospects and challenges
journal, July 2010
- Rao, Shilpakala Sainath; Mohan, Ketha V. K.; Atreya, Chintamani D.
- Journal of Microbiological Methods, Vol. 82, Issue 1
Anthrax inhalation and lethal human infection
journal, February 2002
- Peters, Cj; Hartley, Dm
- The Lancet, Vol. 359, Issue 9307
Distinctness of spore and vegetative cellular fatty acid profiles of some aerobic endospore-forming bacilli
journal, February 2000
- Song, Yajun; Yang, Ruifu; Guo, Zhaobiao
- Journal of Microbiological Methods, Vol. 39, Issue 3
Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation
journal, March 2011
- Rasko, D. A.; Worsham, P. L.; Abshire, T. G.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 12
DINAMelt web server for nucleic acid melting prediction
journal, July 2005
- Markham, N. R.; Zuker, M.
- Nucleic Acids Research, Vol. 33, Issue Web Server
Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis---One Species on the Basis of Genetic Evidence
journal, June 2000
- Helgason, E.; Okstad, O. A.; Caugant, D. A.
- Applied and Environmental Microbiology, Vol. 66, Issue 6
Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes
journal, September 2006
- Hoffmaster, A. R.; Hill, K. K.; Gee, J. E.
- Journal of Clinical Microbiology, Vol. 44, Issue 9
Bacillus thuringiensis subsp.konkukian (Serotype H34) Superinfection: Case Report and Experimental Evidence of Pathogenicity in Immunosuppressed Mice
journal, January 1998
- Hernandez, Eric; Ramisse, Francoise; Ducoureau, Jean-Pierre
- Journal of Clinical Microbiology, Vol. 36, Issue 7
Anthrax Toxin: Receptor Binding, Internalization, Pore Formation, and Translocation
journal, June 2007
- Young, John A. T.; Collier, R. John
- Annual Review of Biochemistry, Vol. 76, Issue 1
What Sets Bacillus anthracis Apart from Other Bacillus Species?
journal, October 2009
- Kolstø, Anne-Brit; Tourasse, Nicolas J.; Økstad, Ole Andreas
- Annual Review of Microbiology, Vol. 63, Issue 1
A microbial detection array (MDA) for viral and bacterial detection
journal, January 2010
- Gardner, Shea N.; Jaing, Crystal J.; McLoughlin, Kevin S.
- BMC Genomics, Vol. 11, Issue 1
A Functional Gene Array for Detection of Bacterial Virulence Elements
journal, May 2008
- Jaing, Crystal; Gardner, Shea; McLoughlin, Kevin
- PLoS ONE, Vol. 3, Issue 5
Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing
journal, August 2010
- Chen, Peter E.; Willner, Kristin M.; Butani, Amy
- PLoS ONE, Vol. 5, Issue 8
Investigation of Bioterrorism-Related Anthrax, United States, 2001: Epidemiologic Findings
journal, October 2002
- Jernigan, Daniel B.; Raghunathan, Pratima L.; Bell, Beth P.
- Emerging Infectious Diseases, Vol. 8, Issue 10
Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen
journal, October 2002
- Quinn, Conrad P.; Semenova, Vera A.; Elie, Cheryl M.
- Emerging Infectious Diseases, Vol. 8, Issue 10
Sverdlovsk Revisited: Pulmonary Pathology of Inhalational Anthrax Versus Anthraxlike Bacillus cereus Pneumonia
journal, March 2012
- Walker, David
- Archives of Pathology & Laboratory Medicine, Vol. 136, Issue 3
Works referencing / citing this record:
Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages
journal, April 2016
- Sharp, Natasha J.; Molineux, Ian J.; Page, Martin A.
- Applied and Environmental Microbiology, Vol. 82, Issue 8
Targeted amplification for enhanced detection of biothreat agents by next-generation sequencing
journal, November 2015
- Gardner, Shea N.; Frey, Kenneth G.; Redden, Cassie L.
- BMC Research Notes, Vol. 8, Issue 1
Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis
journal, February 2019
- Thissen, James B.; Be, Nicholas A.; McLoughlin, Kevin
- PLOS ONE, Vol. 14, Issue 2
Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood
journal, January 2014
- Frey, Kenneth G.; Herrera-Galeano, Jesus; Redden, Cassie L.
- BMC Genomics, Vol. 15, Issue 1
Identification of Bacillus and Yersinia species and hoax agents by protein profiling using microfluidic capillary electrophoresis with peak detection algorithms
journal, July 2019
- Bowman, Sorelle; Casares-de-Cal, María-Ángeles; Alvarez-Dios, Jose
- Australian Journal of Forensic Sciences, Vol. 53, Issue 1
Metagenomic Analysis of the Airborne Environment in Urban Spaces
journal, October 2014
- Be, Nicholas A.; Thissen, James B.; Fofanov, Viacheslav Y.
- Microbial Ecology, Vol. 69, Issue 2
Temporal dynamics in microbial soil communities at anthrax carcass sites
journal, September 2017
- Valseth, Karoline; Nesbø, Camilla L.; Easterday, W. Ryan
- BMC Microbiology, Vol. 17, Issue 1
Targeted amplification for enhanced detection of biothreat agents by next-generation sequencing
journal, November 2015
- Gardner, Shea N.; Frey, Kenneth G.; Redden, Cassie L.
- BMC Research Notes, Vol. 8, Issue 1
Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis
journal, February 2019
- Thissen, James B.; Be, Nicholas A.; McLoughlin, Kevin
- PLOS ONE, Vol. 14, Issue 2