Detection of <i>Bacillus anthracis</i> DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

<div><p><i>Bacillus anthracis</i> 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 <i>B. anthracis</i> genome, however, renders specific detection difficult, due to close homology with <i>B. cereus</i> and <i>B. thuringiensis</i>. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of <i>B. anthracis</i> in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of <i>B. anthracis</i> 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 <i>B. anthracis</i> 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 <i>B. anthracis</i> could be reliably distinguished from sequence data mapping to either <i>B. cereus</i> or <i>B. thuringiensis</i>. We also demonstrated the efficacy of a microbial census microarray in detecting <i>B. anthracis</i> 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.</p> </div

Mapping of Illumina reads to closely related <i>Bacillus</i> species.

<p>Following sequencing of <i>B. anthracis</i>-spiked environmental samples, mapping specificity was examined by determining the percent of total Illumina reads mapping to the closely related species <i>B. thuringiensis</i> Al Hakam and <i>B. cereus</i> biovar <i>anthracis</i> CI. Illumina reads were obtained from A. aerosol background DNA and <b>B</b>. soil background DNA samples spiked with increasing amounts of <i>B. anthracis</i> DNA. </p

IS element found in NE061598 compared to Schu S4.

<p>IS element found in NE061598 compared to Schu S4.</p

Alignment of unique 454 reads to <i>B. anthracis</i> and near-neighbor species.

<p>454 reads mapping only to <i>B. anthracis</i> or a close relative, discounting reads mapping to multiple reference genomes, were identified. The number of sequencing reads mapping uniquely to <i>B. anthracis</i> Ames DNA are shown compared to <b>A</b>. <i>B. thuringiensis</i> in aerosol samples, <b>B</b>. <i>B. thuringiensis</i> in soil samples, <b>C</b>. <i>B. cereus</i> in aerosol samples, and <b>D</b>. <i>B. cereus</i> in soil samples. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073455#pone-0073455-g004" target="_blank">Figure 4</a>, this analysis was performed separately for each set of species.</p

Alignment of uniquely mapped Illumina reads to genomes from <i>B. anthracis</i> and two closely-related species.

<p>Due to the high degree of sequence similarity among the three examined <i>Bacillus</i> species, a unique mapping approach was used. DNA sequencing reads mapping to only <i>B. anthracis</i> or one of the two near neighbor species were identified; reads mapping to multiple reference genomes were ignored. This approach facilitated distinction among the three closely related species. The number of uniquely mapped reads for <i>B. anthracis</i> is given compared to <b>A</b>. <i>B. thuringiensis</i> in aerosol samples, <b>B</b>. <i>B. thuringiensis</i> in soil samples, <b>C</b>. <i>B. cereus</i> in aerosol samples, and <b>D</b>. <i>B. cereus</i> in soil samples. This analysis was performed separately for each species – unique reads between <i>B. anthracis</i> and <i>B. thuringiensis</i> were identified, followed by identification of unique reads between <i>B. anthracis</i> and <i>B. cereus</i> – thus the results of each comparison are shown in separate charts.</p

Mapping of sequencing reads obtained from <i>B. anthracis</i>-spiked environmental samples to specified reference genomes.

<p><i>B. anthracis</i> Ames genomic DNA was combined with background nucleic acid extracted from either aerosol or soil-based material. Increasing genome copy numbers were spiked into samples at 10-fold concentration intervals. Samples were then subjected to whole genome amplification and next-generation sequencing. The resultant reads were mapped to either a target set (<i>B. anthracis</i> and plasmids) or a background set of DNA sequences, intended to assess non-specific alignment of <i>B. anthracis</i>-derived sequence reads to other genomes. The numbers of reads mapped were normalized to total reads obtained for each sample to standardize results. Shown are the percentage of reads mapped for <b>A</b>. Illumina reads from an aerosol background spiked with <i>B. anthracis</i> genomic DNA, <b>B</b>. Illumina reads from a soil background spiked with <i>B. anthracis</i> DNA, <b>C</b>. 454 reads from an aerosol background spiked with <i>B. anthracis</i> DNA, and <b>D</b>. 454 reads from a soil background spiked with <i>B. anthracis</i> DNA.</p

Genome rearrangement representation for NE061598, Schu S4 and FSC033 genomes.

<p>Each local collinear blocks (LCB) 1–10 is represented by a different color. Upside-down blocks (i.e. LCBs 3 and 9) represent the location of the reverse strand, which means an inversion has occurred. Each LCB is denoted above NE061598.</p

Genome rearrangement representation for NE061598 and Schu S4 genomes.

<p>Each local collinear blocks (LCB) 1-6 is represented by a different color. Upside-down blocks (i.e. LCB2) represent the location of the reverse strand, which means an inversion has occurred. Note the rearrangements of LCB4 and LCB5.</p

Genomic characteristics of <i>F. tularensis</i> subsp. <i>tularensis</i> NE061598.

<p>Genomic characteristics of <i>F. tularensis</i> subsp. <i>tularensis</i> NE061598.</p

VNTR markers and their differences between Schu S4 and NE061598.

a<p>FtM1-FtM25 VNTR markers as previously reported by Johansson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009007#pone.0009007-Johansson1" target="_blank">[7]</a>. New VTNR polymorphisms identified in this study are listed as VNTR1 through VNTR-5.</p>b<p>Indicates repeat size in nucleotides.</p>c<p>“G” indicates that the repeat is located within an open reading frame (genic) whereas “I” indicates that the repeat is located within an intergenic region. Distance to predicted translation start site is indicated in nucleotides. “+” or “−” indicates that the translation start site is downstream or upstream of repeat motif, respectively (as reported by Johansson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009007#pone.0009007-Johansson1" target="_blank">[7]</a>).</p