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Background  Genome-wide analysis of sequence divergence among species offers profound insights into the evolutionary processes that shape lineages. When full-genome sequencing is not feasible for a broad comparative study, we propose the use of array-based comparative genomic hybridization (aCGH) in order to identify orthologous genes with high sequence divergence. Here we discuss experimental design, statistical power, success rate, sources of variation and potential confounding factors. We used a spotted PCR product microarray platform from  Drosophila melanogaster  to assess sequence divergence on a gene-by-gene basis in three fully sequenced heterologous species ( D. sechellia ,  D. simulans , and  D. yakuba ). Because complete genome assemblies are available for these species this study presents a powerful test for the use of aCGH as a tool to measure sequence divergence.    Results  We found a consistent and linear relationship between hybridization ratio and sequence divergence of the sample to the platform species. At higher levels of sequence divergence (< 92% sequence identity to  D. melanogaster ) ~84% of features had significantly less hybridization to the array in the heterologous species than the platform species, and thus could be identified as "diverged". At lower levels of divergence (≥ 97% identity), only 13% of genes were identified as diverged. While ~40% of the variation in hybridization ratio can be accounted for by variation in sequence identity of the heterologous sample relative to  D. melanogaster , other individual characteristics of the DNA sequences, such as GC content, also contribute to variation in hybridization ratio, as does technical variation.    Conclusions  Here we demonstrate that aCGH can accurately be used as a proxy to estimate genome-wide divergence, thus providing an efficient way to evaluate how evolutionary processes and genomic architecture can shape species diversity in non-model systems. Given the increased number of species for which microarray platforms are available, comparative studies can be conducted for many interesting lineages in order to identify highly diverged genes that may be the target of natural selection.

Using comparative genomic hybridization to survey genomic sequence divergence across species: a proof-of-concept from Drosophila