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Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis.

Larsson P, Svensson K, Karlsson L, Guala D, Granberg M, Forsman M, Johanssont A - Emerging Infect. Dis. (2007)

Bottom Line: To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA).MLVA included sequences with much diversity in copy number of tandem repeats.The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

View Article: PubMed Central - PubMed

Affiliation: Swedish Defence Research Agency, Umeå, Sweden.

ABSTRACT
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

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Related in: MedlinePlus

Heat map of marker states for 38 insertion-deletion (indel) and 25 multilocusvariable-number tandem repeat analysis (MLVA) loci examined. Each Francisellatularensis strain is represented by a single row of colored boxes and eachDNA loci by a single column. Relative genetic similarity is represented by thesimilarity of the colors on the gradient scale ranging from blue to yellow. For thebinary indel markers, the state of each marker in the genome of strain F.tularensis subsp. novicida U112 represents the index andis depicted in yellow. Blue indicates the amplification of an allelic variant distinctfrom that of the index genome. For strain ATCC 6223, both alleles were amplified atloci Ftind-32, and the corresponding box is thus divided into a yellow and a bluepart. For MLVA loci, blue represents the largest allele size for each multistatemarker; yellow represents the smallest.
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Figure 3: Heat map of marker states for 38 insertion-deletion (indel) and 25 multilocusvariable-number tandem repeat analysis (MLVA) loci examined. Each Francisellatularensis strain is represented by a single row of colored boxes and eachDNA loci by a single column. Relative genetic similarity is represented by thesimilarity of the colors on the gradient scale ranging from blue to yellow. For thebinary indel markers, the state of each marker in the genome of strain F.tularensis subsp. novicida U112 represents the index andis depicted in yellow. Blue indicates the amplification of an allelic variant distinctfrom that of the index genome. For strain ATCC 6223, both alleles were amplified atloci Ftind-32, and the corresponding box is thus divided into a yellow and a bluepart. For MLVA loci, blue represents the largest allele size for each multistatemarker; yellow represents the smallest.

Mentions: A graphic representation of the observed amplification patterns at indel and MLVA loci isshown in Figure 3. A difference in mutationalstability was apparent between indel and MLVA loci. Indel loci showed a binary patternthat grouped F. tularensis in agreement with traditional taxonomy basedon phenotype. In accordance with previous genetic typing by MLVA, PFGE, or sequencing of 7housekeeping genes, the indel analysis distinguished 2 major subpopulations of type Astrains (denoted A.I and A.II) and also showed Japan-derived F.tularensis strains to be distinct from strains of F. tularensissubsp. holarctica isolated in other parts of the Northern Hemisphere.Furthermore, indel analysis identified additional subpopulations among F.tularensis subsp. holarctica strains. Geographic origins ofthese subpopulations suggest dispersal over large distances. Two strains from the UnitedStates, OSU18 (represented by genome sequence data only) and FSC035, were identical at allindel loci and constitute a distinct genetic entity. Strains FSC012 from the United Statesand FSC519 from Sweden formed another entity. Finally, 6 strains originating in Sweden orRussia represented a third subpopulation. Compared with indel analysis, MLVA showed muchmore extensive polymorphisms, which was helpful for characterizing individual strains.Simpson’s index of diversity ranged between 0.17 and 0.97 for the MLVA loci andbetween 0.09 and 0.52 for the indel loci, which reflects the fact that only 2 allelestates were present for the indel loci while the MLVA loci were more diverse, with up to16 alleles (for MLVA marker Ft-M3).


Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis.

Larsson P, Svensson K, Karlsson L, Guala D, Granberg M, Forsman M, Johanssont A - Emerging Infect. Dis. (2007)

Heat map of marker states for 38 insertion-deletion (indel) and 25 multilocusvariable-number tandem repeat analysis (MLVA) loci examined. Each Francisellatularensis strain is represented by a single row of colored boxes and eachDNA loci by a single column. Relative genetic similarity is represented by thesimilarity of the colors on the gradient scale ranging from blue to yellow. For thebinary indel markers, the state of each marker in the genome of strain F.tularensis subsp. novicida U112 represents the index andis depicted in yellow. Blue indicates the amplification of an allelic variant distinctfrom that of the index genome. For strain ATCC 6223, both alleles were amplified atloci Ftind-32, and the corresponding box is thus divided into a yellow and a bluepart. For MLVA loci, blue represents the largest allele size for each multistatemarker; yellow represents the smallest.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874433&req=5

Figure 3: Heat map of marker states for 38 insertion-deletion (indel) and 25 multilocusvariable-number tandem repeat analysis (MLVA) loci examined. Each Francisellatularensis strain is represented by a single row of colored boxes and eachDNA loci by a single column. Relative genetic similarity is represented by thesimilarity of the colors on the gradient scale ranging from blue to yellow. For thebinary indel markers, the state of each marker in the genome of strain F.tularensis subsp. novicida U112 represents the index andis depicted in yellow. Blue indicates the amplification of an allelic variant distinctfrom that of the index genome. For strain ATCC 6223, both alleles were amplified atloci Ftind-32, and the corresponding box is thus divided into a yellow and a bluepart. For MLVA loci, blue represents the largest allele size for each multistatemarker; yellow represents the smallest.
Mentions: A graphic representation of the observed amplification patterns at indel and MLVA loci isshown in Figure 3. A difference in mutationalstability was apparent between indel and MLVA loci. Indel loci showed a binary patternthat grouped F. tularensis in agreement with traditional taxonomy basedon phenotype. In accordance with previous genetic typing by MLVA, PFGE, or sequencing of 7housekeeping genes, the indel analysis distinguished 2 major subpopulations of type Astrains (denoted A.I and A.II) and also showed Japan-derived F.tularensis strains to be distinct from strains of F. tularensissubsp. holarctica isolated in other parts of the Northern Hemisphere.Furthermore, indel analysis identified additional subpopulations among F.tularensis subsp. holarctica strains. Geographic origins ofthese subpopulations suggest dispersal over large distances. Two strains from the UnitedStates, OSU18 (represented by genome sequence data only) and FSC035, were identical at allindel loci and constitute a distinct genetic entity. Strains FSC012 from the United Statesand FSC519 from Sweden formed another entity. Finally, 6 strains originating in Sweden orRussia represented a third subpopulation. Compared with indel analysis, MLVA showed muchmore extensive polymorphisms, which was helpful for characterizing individual strains.Simpson’s index of diversity ranged between 0.17 and 0.97 for the MLVA loci andbetween 0.09 and 0.52 for the indel loci, which reflects the fact that only 2 allelestates were present for the indel loci while the MLVA loci were more diverse, with up to16 alleles (for MLVA marker Ft-M3).

Bottom Line: To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA).MLVA included sequences with much diversity in copy number of tandem repeats.The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

View Article: PubMed Central - PubMed

Affiliation: Swedish Defence Research Agency, Umeå, Sweden.

ABSTRACT
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

Show MeSH
Related in: MedlinePlus