BMC Genomics 2004, 5:54 http://www.biomedcentral.com/1471-2164/5/54 Table 3: Ambystoma contig search of NCBI human RefSeq, nr, and Xenopus Unigene databases. # BLASTX Best Matches < E-100 < E-50 > E-100 < E-20 > E-50 < E-10 > E-20 < E-7 > E-10 A. mex A. t. tig 6283 630 2015 2153 967 518 5545 870 1990 1595 745 345 A. mexicanum 7909 A. t. tigrinum 6912 2296 523 465 353 X. tropicalis 34296 pVFreigonujnercdetisa2gram of BLAST comparisons among amphibian EST Venn diagram of BLAST comparisons among amphibian EST projects. Values provided are numbers of reciprocal best BLAST hits (E<10-20) among quality masked A. mexicanum and A. t. tigrinum assemblies and a publicly availa- ble X. tropicalis EST assembly http://www.sanger.ac.uk/ Projects/X_tropicalis Identification of vertebrate sequences similar to Ambystoma contigs We searched all contigs against several vertebrate data- bases to identify sequences that exhibited significant sequence similarity. As our objective was to reliably anno- tate as many contigs as possible, we first searched against 19,804 sequences in the NCBI human RefSeq database (Figure 1), which is actively reviewed and curated by biol- ogists. This search revealed 5619 and 4973 "best hit" matches for the A. mexicanum and A. t. tigrinum EST data- sets at a BLASTX threshold of E = 10-7. The majority of contigs were supported at more stringent E-value thresh- olds (Table 3). Non-matching contigs were subsequently searched against the Non-Redundant (nr) Protein data- base and Xenopus tropicalus and X. laevis UNIGENE ESTs (Figure 1). These later two searches yielded a few hundred more 'best hit' matches, however a relatively large number of ESTs from both ambystomatid species were not similar to any sequences from the databases above. Presumably, these non-matching sequences were obtained from the non-coding regions of transcripts or they contain protein- coding sequences that are novel to salamander. Although the majority are probably of the former type, we did iden- tify 3,273 sequences from the non-matching set that had open reading frames (ORFs) of at least 200 bp, and 911 of these were greater than 300 bp. The distribution of ESTs among contigs can provide per- spective on gene expression when clones are randomly sequenced from non-normalized cDNA libraries. In gen- eral, frequently sampled transcripts may be expressed at higher levels. We identified the 20 contigs from A. mexica- num and A. t. tigrinum that contained the most assembled ESTs (Table 4). The largest A. t. tigrinum contigs contained fewer ESTs than the largest A. mexicanum contigs, probably because fewer overall A. t. tigrinum clones were sequenced, with the majority selected from a normalized library. How- ever, we note that the contig with the most ESTs was iden- tified for A. t. tigrinum: delta globin. In both species, transcripts corresponding to globin genes were sampled more frequently than all other loci. This may reflect the fact that amphibians, unlike mammals, have nucleated red blood cells that are transcriptionally active. In addition to globin transcripts, a few other house-keeping genes were identified in common from both species, however the majority of the contigs were unique to each list. Overall, the strategy of sequencing cDNAs from a diverse collection of tissues (from normalized and non-normalized libraries) yielded different sets of highly redundant contigs. Only 25% and 28% of the A. mexicanum and A. t. tigrinum con- tigs, respectively, were identified in common (Figure 2). We also note that several hundred contigs were identified in common between Xenopus and Ambystoma; this will help facilitate comparative studies among these amphibian models. Functional annotation For the 10,592 contigs that showed significant similarity to sequences from the human RefSeq database, we obtained Gene Ontology (23) information to describe ESTs in functional terms. Although there are hundreds of possible annotations, we chose a list of descriptors for molecular and biological processes that we believe are of interest for research programs currently utilizing salaman- ders as model organisms (Table 5). In all searches, we counted each match between a contig and a RefSeq sequence as identifying a different ambystomatid gene, Page 4 of 17 (page number not for citation purposes)