articleA critical assessment of cross-species detection of gene duplicates using comparative genomic hybridization

Background: Comparison of genomic DNA among closely related strains or species is a powerful approach for identifying variation in evolutionary processes. One potent source of genomic variation is gene duplication, which is prevalent among individuals and species. Array comparative genomic hybridization (aCGH) has been successfully utilized to detect this variation among lineages. Here, beyond the demonstration that gene duplicates among species can be quantified with aCGH, we consider the effect of sequence divergence on the ability to detect gene duplicates. Results: Using the X chromosome genomic content difference between male D. melanogaster and female D. yakuba and D. simulans, we describe a decrease in the ability to accurately measure genomic content (copy number) for orthologs that are only 90% identical. We demonstrate that genome characteristics (e.g. chromatin environment and non-orthologous sequence similarity) can also affect the ability to accurately measure genomic content. We describe a normalization strategy and statistical criteria to be used for the identification of gene duplicates among any species group for which an array platform is available from a closely related species. Conclusions: Array CGH can be used to effectively identify gene duplication and genome content; however, certain biases are present due to sequence divergence and other genome characteristics resulting from the divergence between lineages. Highly conserved gene duplicates will be more readily recovered by aCGH. Duplicates that have been retained for a selective advantage due to directional selection acting on many loci in one or both gene copies are likely to be under-represented. The results of this study should inform the interpretation of both previously published and future work that employs this powerful technique. Background It is well established that gene duplication and the subsequent evolution of duplicates is an important source of functional novelty [for review see [1]]. For example, gene duplications are known to be involved in adaptive evolution in response to diet [2-4], chemical challenge [5,6], and reproductive incompatibility [7,8]. Such adaptations can allow diversification into new niches, as has been suggested for cold adaptation [plants: [9], Antarctic ice fish: [10]] novel metabolic processes [C-4 photosynthesis: [11]]. Appreciation for the pervasive nature of gene duplication has been reinforced by genomic studies that identify dramatic variation in gene copy number between individuals and between species [[12], human: [13], mouse: [14,15], comparative mammals: [16], Drosophila: [17], Arabidopsis: [18]]. One genomic technique for identifying gene duplications among lineages is array-based comparative genomic hybridization (aCGH). This technique can identify duplicates that may be collapsed during shotgun sequence assembly [19]. Furthermore, unlike next-generation DNA sequencing technologies, this technique does not rely on a full genome assembly as a reference [[20], e.g. [21]]. In addition to the assessment of copy number variation within a species [e.g. [22]] and between closely related lineages [Drosophila: [23], D. discoideum: [24], experimental evolution in yeast: [25]], array CGH has been applied to the identification of chromosomal aberrations underlying cancer [for review see [26]] and genotyping of individuals within and between populations according to single nucleotide polymorphisms [e.g. Arabidopsis: [27], * Correspondence: renns@reed.edu 1 Department of Biology, Reed College, Portland, OR 97202 USA Full list of author information is available at the end of the article Machado and Renn BMC Genomics 2010, 11:304 http://www.biomedcentral. com/1471-2164/11/304 Page 2 of 9 stickleback fish: [28]]. For each of these applications, when gDNA isolated from one sample is competitively hybridized against gDNA isolated from another sample, genomic regions that have been deleted (or are highly diverged) in the genome of the first sample will fail to hybridize to the array features resulting in a log ratio less than zero. Conversely, genomic regions that have been duplicated in the first sample will hybridize at a ratio of 2:1 (or greater), resulting in a log ratio greater than zero. When using aCGH to compare genome content between two different species, only one of these gDNA samples is isolated from the species for which the microarray was constructed, and the other gDNA sample is isolated from a heterologous species of interest. Through repetition with multiple heterologous species, a phylogenetic study can be performed that can address genome content in a broad range of related organisms. The aCGH technique has been used to reveal genomic regions likely involved in an organism's ability to inhabit a specific environment [Chlamydia trachomatis tissue specificity: [29], Sinorhizobium meliloti root symbiont: [30], Clostridium difficile host specificity: [31]], pathogenicity [[32], Yersinia pesits: [33], Mycobacterium tuberculosis: [34], Vibrio cholerae: [35]], genomic duplications and deletions associated with population divergence and speciation [Anopheles gambiae: [36,37]], and genomic regions that differentiate humans from other primate species [38,39]. Array-based genomic comparisons can also identify orthologs with high sequence divergence because an increased number of basepair differences between the platform species and the heterologous species will cause a detectable reduction in hybridization strength for the heterologous species [40,41]. We have shown a consistent linear relationship between hybridization ratio and sequence divergence. While 40% of the variation in hybridization ratio was accounted for by variation in sequence identity of the heterologous sample to the platform sample, other characteristics of the DNA sequence, such as GC content and alignment characteristics, also contributed to variation in hybridization ratio [40]. The extent to which reduced hybridization due to sequence divergence compromises the ability to accurately identify gene duplications has not been rigorously addressed, nor have the resultant biases in types of gene duplicates been identified. Here we quantify this effect by using genomic content as a model for gene duplication by specifically focusing on X-linked genes, such that these genes are "duplicated" in female individuals (XX) relative to male individuals (XY). Using three Drosophilid species for which complete genome assemblies are available [42], we survey thousands of orthologs over a range of sequence divergence. We quantify the ability to accurately detect increased genomic content of D. simulans and D. yakuba relative to D. melanogaster males using CGH on a D. melanogaster microarray. We find a decreased ability to identify genomic content with increased sequence divergence, suggesting that array CGH will be biased toward the identification of recent duplicates or otherwise conserved duplicates. We further discuss other potential confounding factors that may affect duplicate detection.