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.

FIG. 1.

Whole-genome XhoI map contig of Y. pestis KIM displayed by the DNAStar software package. The outermost color circle is the consensus map generated by Gentig and is built from the underlying maps represented as arcs. These maps were constructed from individual DNA molecules cleaved with XhoI. Congruent restriction fragments shown in the consensus map are denoted by a common color; the color-ordering scheme is random to provide contrast.

FIG. 2.

Example of sequence validation and correction via optical mapping. Orange arrows indicate XhoI restriction sites. Partial IS elements IS1661-1 and IS1661-2 together make an intact IS1661 element, and partial IS elements IS1541-1 and IS1541-2 together make an intact IS1541 element. Contigs 1 and 2 were originally assembled with each bearing an IS element (IS1661 and IS1541, respectively) disrupted by the insertion of a copy of IS100. The in silico sequence contig maps were not consistent with the optical map. Allowing for a homologous recombination event between the two copies of IS100, however, generated two hybridized IS structures, which was confirmed by new contigs (A and B) whose in silico sequence contig maps perfectly matched the optical map.

FIG. 3.

Comparisons of XhoI and PvuII optical map to sequence data. (A and B) Plots of optical map fragment sizes versus the in silico map fragment sizes from finished sequence for XhoI (A) and PvuII (B). The error bars represent the standard deviation of optical map fragment size on the means. (C and D) Plots of the absolute error rates of optical fragment size versus sequence for XhoI (C) and PvuII (D). (E and F) Cumulative histograms of optical map fragments, by size, for XhoI (E) and PvuII (F).

FIG. 4.

Alignments of the XhoI and PvuII consensus optical maps with the corresponding in silico maps. The outer two circles are the alignment of the in silico PvuII map (the outermost circle) with the PvuII optical map, while the inner two circles show the alignment of the in silico XhoI map (the outer one) with the XhoI optical map. Arrows with solid triangular heads denote missing fragments (missing fragments less than 1 kb are not shown); slender arrows denote missing cuts; arrows with diamonds denote false cuts. Blue and red arrows refer to the XhoI and PvuII maps, respectively.

FIG. 5.

XhoI and PvuII composite optical-map alignment with the in silico XhoI and PvuII map. The composite optical map was generated by normalizing the single-enzyme maps to the same size. Both the in silico and optical maps were broken into 500-kb segments and then leftmost aligned (in a pairwise fashion), using the sequence as the guiding template. The green and blue lines represent the in silico and optical maps, respectively. Scale = 100 kb.

FIG. 5.

XhoI and PvuII composite optical-map alignment with the in silico XhoI and PvuII map. The composite optical map was generated by normalizing the single-enzyme maps to the same size. Both the in silico and optical maps were broken into 500-kb segments and then leftmost aligned (in a pairwise fashion), using the sequence as the guiding template. The green and blue lines represent the in silico and optical maps, respectively. Scale = 100 kb.