A Whole-Genome Shotgun Optical Map of Yersinia pestis Strain KIM

Abstract

Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.There are 11 species in the genus Yersinia, 3 of which are pathogenic for humans (Yersinia pestis, Y. enteroclitica, and Y. pseudotuberculosis). Of the three pathogenic species, Y. pestis, which lives in rodents, transmits this pathogen to humans via fleas, causing bubonic, septicemic, and pneumonic plagues (also known as black death). Y. pestis has been responsible for many recurrent devastating pandemics throughout history, resulting in widespread loss of human life (1,7,22). To understand the molecular mechanisms that define the pathogenicity of this bacterial species, sequencing of two strains (KIM and CO-92 biovar Orientalis) of Y. pestis was funded by the National Institute of Allergy and Infectious Diseases and Beowulf Genomics. The two separate sequencing projects were conducted by F. Blattner's laboratory (University of WisconsinMadison) and Sanger Center (Hinxton, United Kingdom), using the strategy of whole-genome shotgun sequencing (11). Although this sequencing strategy has racked up an impressive number of completed microbial genomes, it can be further optimized in terms of the cost and effort required during the finishing stages. In this regard, physical maps serve to guide sequence assembly, characterize gaps, and validate the finished sequence. Furthermore, ordered restriction maps are particularly useful when attempting to assemble genomic regions containing repeats, since cleavage patterns can accurately discern such sequence elements.The costly appellation of "finished," assumed when dealing with high-quality sequence data, minimally mandates that no genomic region be excluded from the final results (23) and thus requires extensive finishing efforts. The chances that entire regions of a genome might be excluded from "completed" sequence increases in the face of limited budgets and the absence of physical mapping data. Because of these issues, optical maps were used (15) at an early stage of sequence assembly to iden...

Genome Rearrangements of Completely Sequenced Strains of Yersinia pestis

“…Unlike the other two human pathogenic species (Y. pseudotuberculosis and Y. enterocolitica), Y. pestis causes infectious bubonic and pneumonic plague in humans that are of great importance to public health and biodefense. It is thought that Y. pestis was responsible for three major pandemics throughout history, taking tens of thousands of lives (3,7,12). Plague is a zoonotic disease spread by rodents and their fleas and has already been classified as a reemerging disease.…”

Comparative Whole-Genome Mapping To Determine Staphylococcus aureus Genome Size, Virulence Motifs, and Clonality

“…paratuberculosis strain K-10, including identification of two new genes. In fact, it has been shown that Whole Genome Mapping could be routinely used as an aid to bacterial genome sequence assembly (11) and closure of sequence gaps (31). Using comparative whole-genome mapping, we (24) have also shown the presence of a large genetic inversion event in a methicillin-resistant S. aureus (MRSA).…”

Fast and Cheap Genome Wide Haplotype Construction via Optical Mapping

“…A brief description of the data generated by Optical Mapping technology is given in the following section, and many more details of the technology can be found in the literature (e.g. [15,18,25]). Unfortunately, the input genomic data that can be collected from a single DNA molecule by the best chemical and optical methods (such as those used in Optical Mapping) are corrupted by many poorly understood noise processes.…”

“…H 2,i , with indices 0 ≤ i ≤ (N + 1), where H 2,0 and H 2,N +1 represent the chromosome ends), but are unknown. However, short DNA fragments of around 500 Kb derived from such maps, and further corrupted by experimental noise processes can be readily generated at high throughput and very low cost using a technology like Optical Mapping [15,18,25]. These short DNA fragments will be written as D k , with indices 1 ≤ k ≤ M , where M is the number of data fragments and each data fragment is in turn represented as a sequence of restriction sites (e.g.…”

“…To check if this is a problem, we generated simulated data approximating the first 5 mega bases of human chromosome 21, and the typical error rates for Optical Mapping data based on maximum likelihood estimates from previous microbial maps [15,18,25] (The more recent data used in the previous sections had maximum likelihood error estimates about 30% better, so the estimates used here are conservative). The simulated data was assembled using different amounts of simulated data corresponding to data redundancy of 6×, 12×, 16×, 24×, 50× and 100× (per haplotype).…”