Comparative Genomics of 2009 Seasonal Plague (Yersinia pestis) in New Mexico

Plague disease caused by the Gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19th century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen

Pathosphere.org: pathogen detection and characterization through a web-based, open source informatics platform

Background The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. Results The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. Conclusions By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available

Genetic Diversity of the 2009 Plague Isolates.

<p>Distribution of mutations through the 2009 isolates. Mutations relative to the parent strain (CO92) are indicated by black squares. Grey squares indicate that the mutation was not called automatically but was evident by manual inspection of the assembly. Mutation 31 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone-0031604-t002" target="_blank">Table 2</a> is not shown nor incorporated into the phylogeny as it is an expansion of a 10 bp repeating sequence.</p

Phylogenetic Analysis of 2009 Plague Isolates.

<p>The phylogenies of the 2009 isolates were inferred from MLVA data (Panels A and B) or the SNP/Indel data (panels C and D). In all cases, the Pestoides F strain was utilized as an outgroup. <b>Panels A and B</b>: Reconstruction of relationships from MLVA data. (<b>A</b>) Neighbor Joining Analysis of MLVA data. Numbers above branches represent NJ analysis bootstrap proportions, greater than 50%, based on 1000 replications; numbers below branches represent MP analysis bootstrap support. (<b>B</b>) Maximum Parsimony analysis. Numbers above branches represent bootstrap proportions, greater than 50%, based on 1000 replications; numbers below branches represent Majority Rule consensus values. <b>Panels C and D</b>: Phylogenetic analysis using newly identified SNPs/Indels using the (<b>C</b>) Maximum Likelihood method. The tree with the highest log likelihood (−411.0996) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. (<b>D</b>) Phylogenetic analysis using the Maximum Parsimony method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Additional details for the methods employed in phylogenetic reconstructions can be found in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#s2" target="_blank">Methods</a>.</p

New Mexico is a Portion of the Enduring North American Plague Focus.

<p>Highly virulent <i>Yersinia pestis</i> can be found among numerous rodent and animal hosts, occasionally infecting humans and their pets when they come into contact with infected animals or fleas. Samples were chosen for sequencing to represent examples coming from each broad geographic region. Common hosts include squirrels, cats, prairie dogs (<i>Cynomys gunnissoni</i>) and rabbits (<i>Sylvilagus audubonii</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone.0031604-Perry1" target="_blank">[3]</a>. Sequenced isolates are shown with red markers; other cases with green markers (Location map on right). Locations of isolation of strains were mapped using Google Earth®. Source: New Mexico State Dept. of Public Health <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone.0031604-New1" target="_blank">[32]</a>.</p

IS Element Variation Between Strains.

<p>*<i>pldA/IS100</i> chimeric reads are present in a subset of 25% of reads that map to this position (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone.0031604.s002" target="_blank">Figure S2</a>). The remainder of the reads corresponded to an intact <i>pldA</i> gene.</p

Identification of New IS Element Insertion Points in 2009 Strains.

<p><b>A</b>) Locations of new IS element insertions. IS element insertions were identified in templated assembly experiments using CO92 as a reference. New insertion points were identified using Newbler's 454HCStructVars.txt file and the identity of the newly inserted element was determined by BLAST analysis of sequence reads containing novel junctions. Shaded square indicates the presence of an IS element beginning at the indicated nucleotide position. <b>B</b>) Phylogenetic analysis of 2009 strains using Maximum Likelihood method. Each insertion was treated as a single character. <b>C</b>) Phylogenetic analysis using Maximum Parsimony method.</p

Mutations Identified in New Mexico <i>Y. pestis</i> Strains.

<p>Mutations Identified in New Mexico <i>Y. pestis</i> Strains.</p

SNP Discovery in New North American <i>Y. pestis</i> Genome Sequences.

<p>The number of strain-specific newly identified SNPs relative to CO92 is plotted for each isolate. Previously sequenced strains are indicated by shaded bars with the location of origin and the number of strains showing identical genotypes in parentheses. ^* Number of newly identified SNPs described in references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone.0031604-Touchman1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031604#pone.0031604-Auerbach1" target="_blank">[11]</a>.</p

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