| Summary: | Enhancers have been described to evolve by permutation without changing function. This has posed the problem of how to predict enhancer elements that are hidden from alignment-based approaches due to the loss of co-linearity. Alignment-free algorithms have been proposed as one possible solution. However, this approach is hampered by several problems inherent to its underlying working principle. Here we present a new approach, which combines the power of alignment and alignment-free techniques into one algorithm. It allows the prediction of enhancers based on the query and target sequence only, no matter whether the regulatory logic is co-linear or reshuffled. To test our novel approach, we employ it for the prediction of enhancers across the evolutionary distance of ~450Myr between human and medaka. We demonstrate its efficacy by subsequent in vivo validation resulting in 82% (9/11) of the predicted medaka regions showing reporter activity. These include five candidates with partially co-linear and four with reshuffled motif patterns. Orthology in flanking genes and conservation of the detected co-linear motifs indicates that those candidates are likely functionally equivalent enhancers. In sum, our results demonstrate that the proposed principle successfully predicts mutated as well as permuted enhancer regions at an encouragingly high rate.
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