Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 [63,71]. To detect whether footprints are expressed and thus may be ncRNA genes, a blastn search of all foot- prints against the NCBI database of ESTs was per- formed. Footprints with one or more EST matches were screened against the Functional RNA Database [49] for similarity with known ncRNAs. In addition, the RNAz server (Vienna RNA server, http://rna.tbi.univie.ac.at) was used to identify thermodynamically stable and evo- lutionarily conserved RNA secondary structures in the footprint alignments. A Dollo parsimony reconstruction of the genes and footprints was done with MacClade v4.06 [72] to assess whether putative cis-regulatory elements and ncRNAs were lost or gained during vertebrate evolution. Additional material Additional file 1: Summary of the conserved sequences in vertebrate Hox clusters. This table gives an overview of the positions of the coding regions in the vertebrate sequences as well as all footprint positions identified by the tracker software. The footprints are named fp1 to fp33, and footprints that are expressed in at least two species are indicated in bold. Additional file 2: EST blast hits of expressed footprints. All ESTs that correspond to the expressed footprints are given with Genbank accession number, species name and tissue source. Acknowledgements We thank Joel Sohn for help with the caecilians, Andrew Stuart and the other members of the Amemiya Lab for technical assistance and Aru for genome size measurements. We are grateful to Kim Roelants for the drawings and to Wolfgang Otto for help with the tracker software. AM was supported by a Travel Grant of the Research Foundation - Flanders (FWO) for her stay in Seattle. FB and AM received financial support from the Vrije Universiteit Brussel (Concerted Research Action 29). 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