Genome Sequence of Francisella tularensis subspecies holarctica Strain FSC200, Isolated from a Child with Tularemia

Abstract

Here we report the complete, accurate 1.89-Mb genome sequence of Francisella tularensis subsp. holarctica strain FSC200, isolated in 1998 in the Swedish municipality Ljusdal, which is in an area where tularemia is highly endemic. This genome is important because strain FSC200 has been extensively used for functional and genetic studies of Francisella and is well-characterized.

GENOME ANNOUNCEMENT

Strain FSC200 (Francisella Strain Collection, Swedish Defense Research Agency, Umeå, Sweden) was obtained in 1998 during an outbreak of tularemia with 88 human cases between late July and late September in Ljusdal, Sweden (22). Strain FSC200 infected a 4-year-old girl who fell ill with an influenza-like illness and a tender inguinal gland (7). The girl developed a body temperature of >40°C and a 3-mm skin pustule above the right knee. A swab sample taken from the pustule was sent for microbial culture with a clinical suspicion of tularemia. Francisella tularensis was isolated from the swab, and a stock culture was made without any further passages before long-term storage at −80°C. Strain FSC200 has been shown to be fully virulent in mice and is genetically characterized as F. tularensis subsp. holarctica genetic clade B.22 (5, 23, 24).

Here, we present the complete genome sequence of F. tularensis strain FSC200 that was obtained using Sanger and Illumina sequencing protocols and data collection tools. The DNA was from the first culture passage of the original FSC200 stock. A total of 41,127 paired-end Sanger sequence reads from randomly picked small-insert plasmid clones and 960 paired-end sequence reads from randomly picked fosmid clones were collected. The sequences were assembled and viewed using phred/phrap/consed software and provided 12-fold coverage (3, 4). The genome underwent five rounds of autofinish experiments and a final manual inspection. Finishing strategies also included targeted finishing experiments, PCR amplifications and sequencing, and transposon mutagenesis followed by sequencing of selected small-insert plasmid clones and shotgun sequencing of select fosmid clones. The final assembly was validated by using the GenVal software (http://depts.washington.edu/uwgcmed/software.html) (6) developed to compare the virtual-sequence-derived fingerprint pattern of the final assembly with the experimentally derived fingerprint pattern from the fosmid clones. A complete correspondence of the fingerprint patterns provided a kilobase range validation of the final assembly (19). The final Sanger genome sequence was further certified by successful mapping of 6,234,727 paired-end reads from an Illumina HiSeq2000 machine (Illumina Inc., San Diego, CA) to the sequence. Genome annotation was performed by the Prokaryotic Genome Analysis Tool (PGAT) (http://tools.nwrce.org/pgat/) (1).

The completed genome sequence of strain FSC200 consists of 1,894,157 bases with a GC content of 32.2%. Annotation by PGAT predicted 1,442 functional coding sequences (CDSs) and 323 pseudogenes. The 1,442 CDSs have an average length of 311 bp; consequently, 70.9% of the base pairs are coding sequence. There are 38 tRNAs and 10 rRNAs predicted. The FSC200 genome contains 107 insertion sequence elements.

This genome sequence is of great importance due to a well-characterized origin combined with well-characterized virulence properties. Strain FSC200 has served as a reference strain in numerous recent studies (e.g., 2, 5, 8–13, 15–18, 20–23, 25–28).

Nucleotide sequence accession numbers.

The whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank (BioProject PRJNA16087) under accession number AASP00000000. The first version was assigned accession number AASP01000000 and consists of 39 contigs (AASP01000001 to AASP01000039). This version, the second version, has been deposited under accession number CP003862 and consists of the complete genome. The sequence and annotation data are also available on the PGAT website (http://tools.nwrce.org/pgat/) (1) and on the Integrated Microbial Genome Expert Review (IMG/ER) website (https://img.jgi.doe.gov/er/) (14). The Sequence Reads Archives (SRAs) has been deposited under accession numbers SRS348400 (sample), SRX157666 (experiment), and SRR518502 (run).