Dataset Information

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 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.

SUBMITTER: Be NA

PROVIDER: S-EPMC3767809 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

Be Nicholas A NA Thissen James B JB Gardner Shea N SN McLoughlin Kevin S KS Fofanov Viacheslav Y VY Koshinsky Heather H Ellingson Sally R SR Brettin Thomas S TS Jackson Paul J PJ Jaing Crystal J CJ

PloS one 20130909 9

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 elec ...[more]

PMID: 24039948

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Project description:BACKGROUND:In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. RESULTS:To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare false-positive SNPs were associated with variable nucleotide tandem repeats. CONCLUSIONS:The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution.

Dataset Information

Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

Publications

Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

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