Evaluation of one- and two-color gene expression arrays for microbial comparative genome hybridization analyses in routine applications

Abstract

DNA microarray technology has already revolutionized basic research in infectious diseases, and whole-genome sequencing efforts have allowed for the fabrication of tailor-made spotted microarrays for an increasing number of bacterial pathogens. However, the application of microarrays in diagnostic microbiology is currently hampered by the high costs associated with microarray experiments and the specialized equipment needed. Here, we show that a thorough bioinformatic postprocessing of the microarray design to reduce the amount of unspecific noise also allows the reliable use of spotted gene expression microarrays for gene content analyses. We further demonstrate that the use of only single-color labeling to halve the costs for dye-labeled nucleotides results in only a moderate decrease in overall specificity and sensitivity. Therefore, gene expression microarrays using only single-color labeling can also reliably be used for gene content analyses, thus reducing the costs for potential routine applications such as genome-based pathogen detection or strain typing.

FIG. 1.

Comparison of spot intensities (upper panels) and the corresponding densities (lower panels) between one- and two-color arrays. The two-color arrays (left panels) have clearly a wider spread, lower overlap, and therefore better differentiation between absent or present genes. Note that the absence or presence data in the first plots (upper panels) are binary values that have been jittered solely for visualization purposes.

FIG. 2.

ROC curves for one- and two-color arrays. ROC curves are shown for both one-color (top left) and two-color (bottom left) arrays for all six strains (colored) and the complete data set (black), which was used for determination of the optimal threshold (right). The threshold fitted on the original data set had its optimum at 10.6341 at an error rate of 0.0182 in the two-color case and 10.2842 at 0.0565 in the one-color case. err, error.

FIG. 3.

Gene absence and presence comparisons for six meningococcal strains. Venn diagrams of gene presence comparing genome sequencing with one-color and two-color arrays over all six test strains are shown. For the vast majority of the genes (92.94, 92.26, 91.61, 93.15, 91.14, and 94.09% from left to right, top to bottom) for genome sequencing, the one- and two-color arrays were in agreement (total intersection set and universe). Prediction errors accumulate in the intersections, where one-color predictions contradict both two-color and genome analyses (66 and 19 in α14, 52 and 13 in α153, 30 and 13 in α275, 56 and 17 in α710, 79 and 39 in MC58, and 48 and 24 in Z2491).