Discriminatory power and linkage disequilibrium analysis results for 5 sets of VNTR markers: the newly proposed MLVA7 and MLVA8, MLVA15, MLVA25, MLVA31.

<p>*Values as calculated from MLVA31 data published by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Beyer1" target="_blank">[16]</a>.</p

(a) Minimum spanning tree of MLVA31 data from 119 animal and environmental <i>B. anthracis</i> strains.

<p>Each circle represents a unique genotype. The diameter of each circle corresponds to the number of isolates with the same genotype. Genotypes connected by a shaded background differ by a maximum of 3 of the 31 VNTR markers and could be considered as a “clonal complex”. Thick connecting lines represent one locus differences; regular connecting lines represent two loci differences. The length of each branch is proportional to the number of differences. Each epidemiological situation is represented by a specific color as defined in part b.(<b>b</b>). Localization of the 119 animal and environmental <i>B. anthracis</i> strains.</p

Congruence analysis of 5 sets of VNTR markers: MLVA7, MLVA8, MLVA15, MLVA25 and MLVA31.

<p>a congruence analysis in the “France” dataset (this study); b congruence analysis in the “Namibia” dataset <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Beyer1" target="_blank">[16]</a>.</p

Geographical repartition of the strains for each clonal complex (CC).

<p>^aCC1 strains. ^bCC2 (black circle), and CC3 (grey circle) strains. ^cCC4 strains.</p

Electrophoresis gel of the MLVA7 panel on four strains.

<p>Well 1: Sterne strain, wells 2 to 4: French bovine strains. Migration on 3% standard agarose gel at 110 V during 4 hours. A 100 bp ladder was used running from 100 bp up to 1000 bp, the 500 bp and 1000 bp bands are more intense.</p

Genotyping of French <i>Bacillus anthracis</i> Strains Based on 31-Loci Multi Locus VNTR Analysis: Epidemiology, Marker Evaluation, and Update of the Internet Genotype Database

<div><p>Background</p><p><i>Bacillus anthracis</i> is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described.</p><p>Results</p><p>MLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site.</p><p>Conclusions</p><p>The present report provides a wide coverage of the genetic diversity of naturally occurring <i>B. anthracis</i> strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as <i>B. anthracis</i>.</p></div

List of 32 published <i>B. anthracis</i> VNTR markers.

a<p>Keim et al. 2000 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Keim2" target="_blank">[6]</a>;</p>b<p>Le Flèche et al. 2001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-LeFlche1" target="_blank">[10]</a>;</p>c<p>Keim et al. 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Keim3" target="_blank">[15]</a>;</p>d<p>Lista et al. 2006 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Lista1" target="_blank">[11]</a>;</p>e<p>Leski et al. 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Leski1" target="_blank">[43]</a>.</p><p>*Locus not currently used due to some large amplicon sizes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-LeFlche1" target="_blank">[10]</a>. The numerical allele coding convention is as published by Keim et al., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Keim2" target="_blank">[6]</a>, Lista et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Lista1" target="_blank">[11]</a> and Antwerpen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131-Antwerpen1" target="_blank">[26]</a>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095131#pone.0095131.s001" target="_blank">Data S1</a> for more details on the coding conventions and comparison with alternative coding conventions.</p

Graphic comparison of the discriminatory power and the contribution to the standardized index of association (I_A^S) for each VNTR marker.

<p>Graphic comparison of the discriminatory power and the contribution to the standardized index of association (I_A^S) for each VNTR marker.</p

Comparison of the diversity index (DI) of individual loci in the “France” versus “Namibia” datasets.

<p>Comparison of the diversity index (DI) of individual loci in the “France” versus “Namibia” datasets.</p

CRISPR genotypes.

<p>CRISPR genotypes.</p