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Abstract
Prefabricated expression microarrays are currently available for only a few species but methods have been proposed to extend their application to comparisons between divergent genomes. Here we demonstrate that the hybridization intensity of genomic DNA is a poor basis on which to select unbiased probes on Affymetrix expression arrays for studies of comparative transcriptomics, and that doing so produces spurious results. We used the Affymetrix Xenopus laevis microarray to evaluate expression divergence between X. laevis, X. borealis, and their F1 hybrids. When data are analyzed with probes that interrogate only sequences with confirmed identity in both species, we recover results that differ substantially analyses that use genomic DNA hybridizations to select probes. Our findings have implications for the experimental design of comparative expression studies that use single-species microarrays, and for our understanding of divergent expression in hybrid clawed frogs. These findings also highlight important limitations of single-species microarrays for studies of comparative transcriptomics of polyploid species.
Figure 1. Genomic hybridization intensities (gDNA intensity) of XL, XB, and XM vary with respect to the non-target to target ratio of these intensities (gDNA ratio).This graph depicts the median gDNA intensities of all probes on the chip ranked by their gDNA ratio into 25 bins; each bin contains 10,000 probes except the 25th bin, which contains 7852 probes. The area in gray corresponds with the range of gDNA ratios of probes that are retained using the method of Malone et al. (2007). XL gDNA ratios are represented by filled symbols and non-target gDNA ratios are represented by unfilled symbols. Shown are relationships between the median gDNA intensity of each bin and the median gDNA ratio of each bin for (A) our XM and XL gDNA hybridizations, (B) the XM and XL gDNA hybridizations of Malone et al. (2007), and (C) our XB and XL gDNA hybridizations.
Figure 2. The gDNA ratio of probes that perfectly match (PM) XL and XB overlaps extensively with the gDNA ratio of probes that mismatch (MM) one species.(A) XB gDNA intensity versus gDNA ratio of PM probes in XL, XL and XB, and XB. PM probes in XL are in black, PM probes in XL and XB are in red, and PM probes in XB but not XL are in green. (B) XB gDNA intensity versus gDNA ratio of MM probes in XB. For comparative purposes, PM probes in XL are again in black. Probes that mismatch both paralogs of genes in XB with one, two, three, or four base pair differences are indicated in red, blue, green, and yellow respectively.
Figure 3. An example of how poor performance of a few probes in the non-target species can affect the rank of many genes, even ones that perform equally in both species.Ten genes (a, b, c, d, e, f, g, h, i, and j) are ranked according to their expression intensity. In the non-target species, probes directed against genes e, h, and j perform poorly and have a low rank in the non-target species due to sequence divergence, even though there actually is no expression divergence. This elevates the rank of many other genes, causing an overall negative median rank difference (RD) and a positively skew in the RD distribution. In this example, significantly upregulated genes in the target species tend to have a higher average rank in this species (9) than the significantly upregulated genes in the non-target species do in that species (6.5). Significantly upregulated genes in the target species have a lower average rank in the non-target species (3) than the significantly upregulated genes in the non-target species do in the target species (3.5).
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