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Title:
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Draft genome sequence of Bacillus anthracis strain Sterne isolate 09RA8929
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Authors:
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Anne Busch1*, Mandy Carolina Elschner1, Daniela Jacob2, Roland Grunow2, Herbert
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Tomaso1
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Germany,
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Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena,
Robert Koch Institute, Centre for Biological Threats and Special Pathogens, Highly
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Pathogenic Microorganisms (ZBS 2), Berlin, Germany
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Corresponding
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[email protected]
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Abstract
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An attenuated Bacillus (B.) anthracis vaccine strain (Sterne) used as an attenuated
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laboratory comparative strain was sequenced and analyzed. A comparison to an
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assembly of B. anthracis strains Sterne isolate (NZ_CP009541 and NZ_CP009540)
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was performed. The lack of the pX02 plasmid and pX01 in approximately five copies
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were confirmed.
author
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Text
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Bacillus (B.) anthracis, a Gram-positive, spore-forming bacillus, is the etiological agent
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of anthrax (1, 2). Anthrax is a zoonotic disease, which can infect humans directly or
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through products. To prevent the livestock of anthrax, vaccines comprising live
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B. anthracis strains (pX01+; pX02−) for animals are available (3). We sequenced and
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analyzed the B. anthracis strain Sterne with the strain collection number 09RA8929 at
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the Friedrich-Loeffler-Institut (FLI), to use it as a laboratory comparative reference
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strain.
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The strain was provided from a repository located at the Robert Koch Institute within
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the framework of the European Union (EU) funded project ‘‘EQADeBa’’ and
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“EMERGE” (Grant Agreement n° 677066) after two passages on Columbia sheep
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blood agar plates (Oxoid, Wesel, Germany). At the FLI the isolate was cultivated for
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diagnostic purposes and to include the isolate in the repository. The strain was
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cultivated twice on Columbia sheep blood agar plates (Oxoid, Wesel, Germany). The
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culture for DNA extraction was initiated from a single colony. DNA was extracted from
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bacterial cells that were grown in cell culture flasks for 24 h. DNA purification was
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performed using QIAGEN Genomic-tip 100/G and QIAGEN Genomic DNA buffer set
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Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Genome
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sequencing was carried out by use of Illumina DNA sequencing at GATC Biotech
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(Germany) with library protocol adapted and validated using a HiSeq sequencer 2500.
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A paired end library was constructed with target insert size of 300 bas pairs resulting
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in 6 million reads. A total of 12 million sequences in a length of 151 bases were
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generated, with an average phred score > 38. The sequences were assembled using
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SPAdes 3.9.1. in Bayes Hammer mode to minimizes mismatches and short indels (--
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careful, (4)) and filtered for contamination with Kraken (5). Subsequently, annotation 2
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was performed with Prokka (in standard settings) (6). The assembly resulted in 33
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contigs with an N50 value of 1162008 and a total sequence length containing
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5366275 bases. Annotation predicted 12 RNAs, 5639 CDS (Coding DNA Sequences),
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79 tRNAs and 1 tmRNA. A phylogenetic analysis on basis of the 16S rRNA sequences
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with Mole-Blast and PhyloPhlAn (7-9) was performed resulting in the consistent results.
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With phylogeny with PhyloPhlAn a differentiation within the Bacillus species was
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possible, although the coding sequences of the plasmids and highly variable
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sequences are excluded with this method (9). This bioinformatics approach is
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complementing the methods of SNP typing and MLVA.
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After quality checking, trimming and merging with bbduk and removal of duplicates the
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reads were mapped to NZ_CP009540, NZ_CP009541 and NZ_CP010794 with the
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bowtie2 module included in geneious (http://www.geneious.com, (7)) and variant
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calling files were generated. Variants are reported with a minimum variant frequency
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of 0.9, with a minimum p value of 10e-60, minimum strand bias p value of 10e-5, and
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supported by at least five reads. A 37-fold mean coverage of the chromosome was
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reported with 5 million bases showing 99.99 % identity with the reference sequence
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(5227150 of 5227496). For the chromosome, 88 variants were called. 36 variants were
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predicted to affect known proteins. The plasmid pX01 yielded a 161-fold mean
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coverage of 181803 bases with 99.8 % identity to the reference sequence (181233 of
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181624). For the plasmid, 13 variants were called; seven of these were affecting known
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proteins (Table 1). With this described method and this data in future Bacillus anthracis
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strains can be monitored. The average coverage ratio chromosome/plasmid suggests
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that the plasmid pX01 is represented with approximately unusual five copies per cell.
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It is reported that sequencing coverage provides exact copy-number estimates for the
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plasmid copy number (Becker et al., 2016). No mapping occurred on pX02 reference
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sequence NZ_CP010794 and the existence of pX02 could be excluded. 3
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Accession
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of B. anthracis strain Sterne isolate 09RA8929 has been deposited in NCBI GenBank
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under the Biosample number SAMN09635715 and the Biosample Number
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PRJNA422985. Raw data are submitted in the Sequence Read Archive (SRA) SRA
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accession: SRP159486.
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Acknowledgements: For this work Anne Busch was supported by a grant of the
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German Federal Ministry of Education and Research and the work was executed within
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the framework of the project Ess-B.A.R. (FKZ 13N13983). We would especially like to
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thank CHAFEA for funding and realizing EMERGE, but also all partners for their
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cooperation and for the external support to the Joint Action by ECDC and DG SANTE.
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References:
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