Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 possible that caecilians and snakes adopted a similar mechanism that resulted in body elongation and limb loss. The distance created between two subsequent genes that are under the control of the same long-range limb enhancer is likely to have some effect on the action of this enhancer on the genes after the void. A number of conserved, non-coding regions have been identified in the HoxD cluster, some of them showing extremely high conservation among all vertebrates. While no new foot- prints arose in the amphibian lineage, at least one, and possibly two conserved sequences were lost. These results, together with data on other vertebrate Hox clus- ters show that tetrapod Hox clusters show more varia- tion than expected previously. Methods Genomic library and DNA sequencing High molecular weight DNA was extracted from ery- throcytes from two Typhlonectes natans specimens and was used to construct a BAC library according to Osoe- gawa et al. [66] and as described by Danke et al. [67]. The genome size was estimated by flow cytometry ana- lysis using chicken erythrocyte nuclei as a standard. EcoRI partial digests of the caecilian DNA were size selected and inserted in the pCC1 BAC vector (Epicen- tre) and the library was combined in 2304 pools con- taining 200 clones each. The library was screened by PCR with primers specific for Hoxd-13 (forward primer: 5’-GCAATGAAGGCGCCTCCAG-3’, reverse primer: 5’- GGAGATATAGGTGTCGTGCCTCGG-3’) to isolate the posterior end of the HoxD cluster. The positive clone was 454 GS FLX-sequenced and assembled by Eurofins MWG Operon (Ebersberg, Germany). The clone was also end sequenced using BigDye 3.1 chemis- try on an ABI 3100 Genetic Analyzer. Several smaller contigs were assembled manually and contig overlaps were confirmed by PCR, except in one case where a gap was present. Because the length of the two contigs is consistent with the size of the insert, we inferred this gap to be small and therefore arbitrarily set it at 100 bp. Caecilian HoxD cluster annotation The genes in the caecilian posterior HoxD cluster were annotated by initial blastx searches of the entire cluster sequence and with the program GenomeScan [31] using mouse HOXD and EVX2 proteins as a training set. Exon - intron boundaries were determined manually by alignment with Hox sequences of other vertebrates. HoxD alignments Global alignments of the caecilian posterior HoxD clus- ter with other vertebrate HoxD clusters were performed with MultiPipMaker [68] and VISTA [69]. The HoxD clusters of the following species were used: Heterodontus Page 7 of 9 francisci (horn shark, AF224263), Danio rerio (zebrafish, UCSC Genome Browser) Latimeria menadoensis (Indonesian coelacanth, FJ497008), Silurana tropicalis (tropical clawed frog, JGI), Gallus gallus (chicken, ENSEMBL), Anolis carolinensis (green anole, UCSC Genome Browser), Monodelphis domestica (grey short- tailed opossum, ENSEMBL), Canis familiaris (domestic dog, ENSEMBL), Homo sapiens (human, NT_005403) and Mus musculus (mouse, AC_015584). The MultiPip- Maker alignments were performed with mouse and T. natans as reference sequence respectively. Interspersed repeats and low complexity regions in all clusters were masked by screening against a library of repetitive elements if available for the organism by RepeatMasker [34]. If no such library was available, as for the horn shark, coelacanth, caecilian, lizard and opossum, the sequences were compared to a database of transposable element encoded proteins. Repeat content The repeat content of the HoxD clusters was deter- mined with Censor [35] and RepeatMasker, using the Repbase library of the species-specific or vertebrate repeats and a database of transposable element encoded proteins. All reported interspersed repeats were taken into account. In addition, self-self blastn analyses were performed to identify direct and inverted repeats within each cluster. Only repeats with maximum 30% mis- match and an e-value ≤ 1e-05 were retained. Analyses of phylogenetic footprints The program tracker [45] was used to detect evolutiona- rily conserved non-coding sequences or phylogenetic footprints. This program is based on blastz [70] to pro- duce initial local pairwise alignments of all pairs of the input sequences. Only the intergenic regions between two homologous genes are compared. After several fil- tering steps, these alignments, which contain a window of 12 nucleotides with minimum identity of 75%, are assembled into groups of partially overlapping regions, resulting in local sequence alignments, or footprint cli- ques. To be able to detect conserved sequences in all non-coding regions, introns were treated as intergenic regions. This analysis was repeated using the same, but repeat masked sequences and all footprints from both analyses were combined. Not every footprint is necessa- rily conserved in each taxon, as its presence or absence may be indicative of the loss of a cis-regulatory element and a subsequent change in gene regulation. Phyloge- netic footprints were treated as two classes: potential (cis-) regulatory elements and putative non-coding RNA genes. Conserved sequences found in the 3’ UTR of genes were screened for the presence of hypothetical target sites of miRNAs using TargetScan Release 5.1