Abstract
Primarily a zoonotic disease, Francisella tularensis is a fastidious intracellular pathogen and is listed as a CDC category A pathogen with notably high pathogenicity. Here we present the scaffolded genome assemblies of nine Francisella strains: eight F. tularensis and one F. philomiragia.
GENOME ANNOUNCEMENT
Francisella tularensis, the causative agent of tularemia (rabbit fever) is considered one of the most pathogenic bacterial infections to humans and has been considered a potential biological weapon for at least the last 70 years (1, 2). Members of this group are small nonmotile, capsule-producing, Gram-negative rods and are often difficult to maintain in laboratory culture.
High-quality genomic DNA was extracted from purified isolates of each strain using QIAgen Genome Tip-500 at USARMIID’s Diagnostic Systems Division (DSD). Specifically, 100-mL bacterial cultures were grown to stationary phase and nucleic acid was extracted per the manufacturer’s recommendations. For BSL3 Francisella, all extracted material was checked for sterility. If sterility was not achieved, the nucleic acid was passed through a 0.45-µM filter and rechecked for viable organisms before removal from the BSL3 suite.
Sequence data for each draft genome was generated using a combination of Illumina and 454 technologies (3, 4). For each genome, we constructed and sequenced an Illumina library of 100-bp reads at high coverage (224- to 1,205-fold genome coverage) and a separate long-insert paired-end library (Roche 454 Titanium platform, 10- to 68-fold genome coverage). The two datasets were assembled together in Newbler (Roche) and the consensus sequences computationally shredded into 2-Kbp overlapping fake reads (shreds). The raw reads were also assembled in Velvet and those consensus sequences computationally shredded into 1.5-Kbp overlapping shreds (5). Draft data from all platforms were then assembled together with ALLPATHS and the consensus sequences computationally shredded into 10-Kbp overlapping shreds (6). We then integrated the Newbler consensus shreds, Velvet consensus shreds, ALLPATHS consensus shreds, and a subset of the long-insert read-pairs using parallel Phrap (High Performance Software, LLC). Possible misassemblies were corrected and some gap closure was accomplished with manual editing in Consed (7–9).
Automatic annotation for each genome utilized an Ergatis-based workflow at LANL with minor manual curation. Each of the nine scaffolded genomes is available in NCBI (accession numbers listed in Table 1) and the raw data can be provided upon request. Preliminary analysis located the genes encoding fopA and tul4 in all of the F. tularensis strains but not in the F. philomiragia strain (as expected). In-depth comparative analyses of these and other genomes are currently underway and will be published in subsequent reports.
TABLE 1.
Basic assembly and annotation statistics
| Strain name | Accession no. (no. of contigs) | Genome size (bp) (% G+C content) | No. of CDSs | No. of rRNAs | No. of tRNAs | Isolation source, yr, location | Alternative ID |
|---|---|---|---|---|---|---|---|
| F. tularensis FAC | JOUF00000000 (44) | 1,847,973 (32.3) | 2,069 | 7 | 36 | Rabbit, 1984, Illinois, USA | DST6755 |
| F. tularensis FAE | JOOR00000000 (9) | 1,854,685 (32.3) | 2,010 | 8 | 36 | Rabbit A, 1988, Illinois, USA | DS88-R-147 |
| F. tularensis Larsen FAF | JOOS00000000 (2) | 1,818,385 (32.3) | 1,980 | 7 | 36 | Unknown, 2006 | NIH38 |
| F. tularensis novicida FAI | JOOT00000000 (25) | 1,970,238 (32.3) | 1,887 | 10 | 38 | Water, 2007, Utah, USA | DPG K4C-IS |
| F. tularensis FTU | JOOU00000000 (22) | 1,864,194 (32.3) | 2,027 | 7 | 36 | Rabbit, 1988, Illinois, USA | DS87-R-135 |
| F. tularensis FTV | JPGP00000000 (96) | 1,887,196 (32.2) | 2,083 | 5 | 34 | Rabbit, 1988, Illinois, USA | DS88-R-160 |
| F. tularensis FTX | JOOV00000000 (27) | 1,880,801 (32.3) | 2,053 | 8 | 36 | Rabbit, 1988, Illinois, USA | DS88-R-144 |
| F. tularensis FTZ | JOVO00000000 (19) | 1,857,471 (32.3) | 5,018 | 7 | 36 | Rabbit, 1987, Illinois, USA | DS87-R-200 |
| F. philomiragia FAJ | JOUE00000000 (6) | 2,050,684 (32.6) | 1,929 | 10 | 39 | Water, 2007, Utah, USA | DPG 3B–W |
Nucleotide sequence accession numbers.
Genome accession numbers to public databases are listed in Table 1.
ACKNOWLEDGMENTS
Funding for this effort was provided by the Defense Threat Reduction Agency’s Joint Science and Technology Office (DTRA J9-CB/JSTO). This manuscript is approved by LANL for unlimited release (LA-UR-14-25133).
The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government.
Footnotes
Citation Davenport KW, Daligault HE, Minogue TD, Bishop-Lilly KA, Broomall SM, Bruce DC, Chain PS, Coyne SR, Frey KG, Gibbons HS, Jaissle J, Koroleva GI, Ladner JT, Palacios GF, Redden CL, Rosenzweig CN, Scholz MB, Teshima H, Johnson SL. 2014. Whole-genome sequences of nine Francisella isolates. Genome Announc. 2(5):e00941-14. doi:10.1128/genomeA.00941-14.
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