Next-generation sequencing of Coccidioides immitis isolated during cluster investigation

Abstract

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source.

Figure 1

Example coverage plot of sequenced genome of Coccidioides immitis. Plot shows base coverage (y-axis) of supercontig 6 from isolate from patient Z, who had coccidioidomycosis. Average depth of coverage for this supercontig was 48.63× over 3,385,806 bases (x-axis) for a total of 164,650,400 bases sequenced.

Figure 2

Alignment of Coccidioides immitis whole-genome sequence reads flanking a confirmed single-nucleotide polymorphism (RSv3 supercontig 1, position 6729646, highlighted in blue in panel B) among the 3 cluster isolates. Isolates from patients X, Y, and Z, who had coccidioidomycosis, are shown in panels A, B, and C, respectively. The alignment was created by using SolScape, a short-read sequence-alignment viewer developed in house (J. Pearson et al., unpub. data; tool available upon request). Reference sequence position is given at the top of each panel; actual reference sequence is highlighted in white at the center of each panel. Bases differing from the reference sequence are highlighted in pink, green, or yellow.

Figure 3

Maximum-parsimony phylogenetic analysis of 13 Coccidioides immitis genomes. MEGA4 (13) was used to conduct maximum-parsimony analysis of all single-nucleotide polymorphism (SNP) loci common to the 3 transplant isolate genomes and the 10 publicly available C. immitis genome sequences (6,10). A total of 32,695 SNP positions were identified in the final dataset, of which 17,080 were parsimony informative. The percentages of replicate trees in which the associated taxa clustered in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale; branch lengths were calculated by using the average pathway method (14) and are in the units of the number of changes over the whole sequence. The consistency index of the tree is 0.63. Scale bar indicates nucleotide substitutions per site.