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Background  Phylogenetic footprinting is an important computational technique for identifying  cis -regulatory motifs in orthologous regulatory regions from multiple genomes, as motifs tend to evolve slower than their surrounding non-functional sequences. Its application, however, has several difficulties for optimizing the selection of orthologous data and reducing the false positives in motif prediction.    Results  Here we present an integrative phylogenetic footprinting framework for accurate motif predictions in prokaryotic genomes (MP 3 ). The framework includes a new orthologous data preparation procedure, an additional promoter scoring and pruning method and an integration of six existing motif finding algorithms as basic motif search engines. Specifically, we collected orthologous genes from available prokaryotic genomes and built the orthologous regulatory regions based on sequence similarity of promoter regions. This procedure made full use of the large-scale genomic data and taxonomy information and filtered out the promoters with limited contribution to produce a high quality orthologous promoter set. The promoter scoring and pruning is implemented through motif voting by a set of complementary predicting tools that mine as many motif candidates as possible and simultaneously eliminate the effect of random noise. We have applied the framework to  Escherichia coli  k12 genome and evaluated the prediction performance through comparison with seven existing programs. This evaluation was systematically carried out at the nucleotide and binding site level, and the results showed that MP 3  consistently outperformed other popular motif finding tools. We have integrated MP 3  into our motif identification and analysis server DMINDA, allowing users to efficiently identify and analyze motifs in 2,072 completely sequenced prokaryotic genomes.    Conclusion  The performance evaluation indicated that MP 3  is effective for predicting regulatory motifs in prokaryotic genomes. Its application may enhance progress in elucidating transcription regulation mechanism, thus provide benefit to the genomic research community and prokaryotic genome researchers in particular.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-016-2982-x) contains supplementary material, which is available to authorized users.

An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes