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      Pyrosequencing Bacillus anthracis

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          Abstract

          Pyrosequencing technology was used to rapidly and specifically identify Bacillus anthracis.

          Abstract

          Pyrosequencing technology is a sequencing method that screens DNA nucleotide incorporation in real time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of light pulses, which produces a profile of characteristic peaks in a pyrogram. We used this technology to identify the warfare agent Bacillus anthracis by sequencing 4 single nucleotide polymorphisms (SNPs) in the rpoB gene as chromosomal markers for B. anthracis. In addition, 1 segment in each of the B. anthracis plasmids pXO1 and pXO2 was analyzed to determine the virulence status of the bacterial strains. Pyrosequencing technology is a powerful method to identify B. anthracis.

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          Anthrax.

          T. Dixon, M Meselson, J. Guillemin … (1999)
          Article 0 comments Cited 161 times – based on 0 reviews     Review now
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            Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing.

            T. Pearson, J Busch, J. RAVEL … (2004)
            Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described "C" branch than to either of two previously described "A" or "B" branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.
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              Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA.

              C Ash, Malcolm Farrow, E Stackebrandt … (1991)
              The primary structures of the 16S rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (greater than 99%) that were consistent with the close relationships shown by previous DNA hybridization studies. The sequences of B. anthracis Sterne and B. cereus emetic strain NCTC 11143 were found to be identical for a continuous stretch of 1,446 bases and differed from the sequence of B. cereus NCDO 1771T (T = type strain) by only a single nucleotide. The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by four to nine nucleotides.
              Article 0 comments Cited 61 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Emerg Infect Dis
                Journal ID (iso-abbrev): Emerging Infect. Dis
                Journal ID (publisher-id): EID
                Title: Emerging Infectious Diseases
                Publisher: Centers for Disease Control and Prevention
                ISSN (Print): 1080-6040
                ISSN (Electronic): 1080-6059
                Publication date (Print): October 2005
                Volume: 11
                Issue: 10
                Pages: 1527-1531
                Affiliations
                [* ]Swedish Institute for Infectious Disease Control, Solna, Sweden
                Author notes
                Address for correspondence: Lars Engstrand, Section of Bacteriology, Swedish Institute for Infectious Disease Control, SE 171 82, Solna, Sweden; fax: 46-8-30-17-97; email: Lars.Engstrand@ 123456smi.ki.se
                Article
                Publisher ID: 04-1316
                DOI: 10.3201/eid1110.041316
                PMC ID: 3366734
                PubMed ID: 16318691
                SO-VID: e62d56e5-c0a4-4e82-ad40-a85fc3551589
                History
                Categories
                Subject: Research
                Subject: Research

                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: bacillus anthracis,pyrosequencing,research,species verification,rpob
                Data availability:
                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: bacillus anthracis, pyrosequencing, research, species verification, rpob

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