BMC Genomics 2004, 5:54 http://www.biomedcentral.com/1471-2164/5/54 tags for two model salamander species (A. mexicanum and A. t. tigrinum). An approximately equivalent number of contigs were identified for each species, with 21,091 unique contigs identified overall. The strategy to sequence cDNAs from a diverse collection of tissues from normal- ized and non-normalized libraries yielded different sets of highly redundant contigs. Only 25% and 28% of the A. mexicanum and A. t. tigrinum contigs, respectively, were identified in common. To demonstrate the utility of these EST resources, we searched databases to identify new probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human/Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander spe- cies. Over 100 new probes were identified for regenera- tion research using informatic approaches. With respect to comparative mapping, 13 of 15 EST markers were mapped successfully, and 6 EST markers were mapped to a previously defined synteny group in Ambystoma. These results indicate a high probability of mapping success using EST markers developed from the joint assembly of A. mexicanum and A. t. tigrinum contigs. Finally, we found that primer sets designed for A. mexicanum / A. t. tigrinum EST orthologues can be used to amplify the corresponding sequence in a related A. tigrinum complex species. Overall, the EST resources reported here will enable a diversity of new research areas using ambystomatid salamanders. Methods cDNA library construction Ten cDNA libraries were constructed for the project using various larval tissues of A. mexicanum and A. t. tigrinum (Table 1). Larval A. mexicanum were obtained from adult animals whose ancestry traces back to the Axolotl Colony [17]. Larval A. t. tigrinum were obtained from Charles Sul- livan Corp. The GARD and MATH A. mexicanum limb regeneration libraries were constructed using regenerating forelimb mesenchyme. Total RNAs were collected from anterior and posterior limbs amputated at the mid-stylo- pod level on 15 cm animals, and from the resulting regenerates at 12 h, 2 days, 5 days and early bud stages. One hundred µg fractions of each were pooled together and polyA-selected to yield 5 µg that was utilized for direc- tional library construction (Lambda Zap, Stratagene). The V1 (A. mex), V2 (A. tig), V4-5 (A. tig), and V6-7 (A. mex) libraries were made from an assortment of larval tissues (see Table 1) using the SMART cDNA cloning kits (Clon- tech). Total RNAs were isolated and reverse transcribed to yield cDNAs that were amplified by long distance PCR and subsequently cloned into pTriplEX. The V3 and AG libraries were constructed by commercial companies (BioS&T and Agencourt, respectively). cDNA template preparation and sequencing cDNA inserts were mass excised as phagemids, picked into microtitre plates, grown overnight in LB broth, and then diluted (1/20) to spike PCR reactions: (94°C for 2 min; then 30 cycles at 94°C for 45 sec, 58°C for 45°sec, and 72°C for 7 min). All successful amplifications with inserts larger than ~500 bp were sequenced (ABI Big Dye or Amersham Dye terminator chemistry and 5' universal primer). Sequencing and clean-up reactions was carried out according to manufacturers' protocols. ESTs were deposited into NCBI database under accession numbers BI817205-BI818091 and CN033008-CN045937 and CN045944-CN069430. EST sequence processing and assembly The PHRED base-calling program [42] was used to gener- ate sequence and quality scores from trace files. PHRED files were then quality clipped and vector/contaminant screened. An in-house program called QUALSCREEN was used to quality clip the ends of sequence traces. Starting at the ends of sequence traces, this program uses a 20 bp sliding window to identify a continuous run of bases that has an average PHRED quality score of 15. Mitochondrial DNA sequences were identified by searching all ESTs against the complete mtDNA genome sequence of A. mex- icanum (AJ584639). Finally, all sequences less than 100 bp were removed. The average length of the resulting ESTs was 629 bp. The resulting high quality ESTs were clustered initially using PaCE [43] on the U.K. HP Superdome com- puter. Multi-sequence clusters were used as input sequence sets for assembly using CAP3 [44] with an 85% sequence similarity threshold. Clusters comprising single ESTs were assembled again using CAP3 with an 80% sequence similarity threshold to identify multi-EST con- tigs that were missed during the initial analysis. This pro- cedure identified 550 additional contigs comprising 1150 ESTs. Functional annotation All contigs and singletons were searched against the human RefSeq database (Oct. 2003 release) using BLASTX. The subset of sequences that yielded no BLAST hit was searched against the non-redundant protein sequence database (Feb. 2004) using BLASTX. The remaining subset of sequences that yielded no BLAST hit was searched against Xenopus laevis and X. tropicalis UNI- GENE ESTs (Mar. 2004) using TBLASTX. Zebrafish ESTs were downloaded from UNIGENE ESTs (May 2004). BLAST searches were done with an E-value threshold of E <10-7 unless specified. Sequence comparison of A. mexicanum and A. t. tigrinum assemblies All low quality base calls within contigs were masked using a PHRED base quality threshold of 16. To identify Page 15 of 17 (page number not for citation purposes)