BMC Genomics 2006, 7:154 http://www.biomedcentral.com/1471-2164/7/154 tors [5'-AATTGGCACGAGG-3', 3'-GCCGTGCTCC-5'], digested with NotI, and directionally cloned into the phagemid vector pT7T3-Pac as described in [17]. Each library comprised several times more recombinants than the expected number of transcripts from the RNA popula- tions utilized, and thus can be treated as if they had the same number of primary recombinants. Sequencing, analysis and clustering Di-deoxy terminator sequencing was performed from the 3' end of the cDNA clones using M13 forward (5'-GTTT- TCCCAGTCAC-3') primers in a 96-well format via cycle sequencing with dRhodamine dye terminator chemistry (Applied Biosystems, Foster City, CA). After thermal cycling, sequencing reactions were processed and ana- lyzed on an ABI-377 or an ABI-3700 capillary sequencer as described in [36]. After data capture on the ABI sequencers, the chromato- graphs are transferred to a centralized server. From there, the sequences were processed as outlined below and placed into our local file-system. Nucleotide sequences and per-base quality values were extracted from the ABI- generated chromatograph files (SCF files) using the Phred-base-calling program and evaluated for 3 features: 1) overall sequence quality (Phred q-score >25); 2) per- cent of sequence (in nt) over q20 > 50%, and 3) the qual- ity-trimmed EST insert length of more than 100 bp [37,38]. ESTprep [39] and RepeatMasker (Smit and Green, unpub- lished data) programs were used to assess the presence of the following EST features: vector cloning site, restriction site, polyadenylation tail and signal sequence, library tag, and potential contaminating sequences from Escherichia coli, V. fischeri and vector as described in [17,40]. Local clustering of the ESTs was performed using the sequence- based clustering program UIcluster [41], allowing matches based on both the forward and reverse comple- ments. A representative sequence was selected from each cluster based on best sequence quality, as described above, resulting in a unigene set containing 13,962 ESTs. Each of the cDNA clones in the unigene set was sequenced from the 5' end using the same protocol described above with the exception of using the M13 reverse primer (5'- AGCGGATAACAATTTCACACAGGA-3') instead of the M13 forward primer. The 3' and 5' sequences were assem- bled into a consensus contig using default settings of the Phred program when sufficient quality sequence existed. 3' and 5' sequences generated as a part of this research were submitted to dbEST division of GenBank at National Center for Biotechnology Information (NCBI) under accession numbers ranging from Genbank:DW251302 to Genbank:DW286722[42]. Sequence annotation Each of the 13,962 ESTs were compared against the six nonredundant peptide sequence databases (GenBank CDS translations, RefSeq Proteins, PDB, SwissProt, PIR, and PRF) available on NCBI using a BLAST-based analysis program (BLASTX), which translates each EST sequence in six reading frames prior to each query. BLAST analyses against peptide sequences provide the largest number of hits in comparison to nucleotide sequences because pep- tide analyses allow for nucleotide sequence divergence among species. Default BLASTX parameters were used including a maximum expectation (E)-value of 10. Because of the low abundance of sequence data from organisms in the sub-kingdom containing mollusks (i.e., Lophotrochozoa) available in NCBI's database, we chose a high E-value threshold to reveal as many potential matches to our squid EST sequences as possible, i.e., sequence annotation was performed using an E-value threshold of e-03 to determine significance. Sequences were first annotated based on a significant hit to an anno- tated protein. If no hit to an annotated protein occurred, then sequences were annotated as a 'significant' or a 'non- significant' 'unknown' (match to a predicted protein sequence from a cDNA or other nondescriptive term) or 'hypothetical' (match to a hypothetical protein). Finally, if the result of the BLAST analysis showed no significant hits to any protein in the NCBI nonredundant database, sequences were annotated as 'no significant hit'. The gene name and a unique identifier (i.e., GI number, Accession Number, Entrez gene ID, Genepept accession) were col- lected from the NCBI web site for each chosen BLAST hit. Putative functions and product information, such as Gene Ontology (GO) for each unique identifier matching an annotated protein was determined with the Database for Annotation Visualization and Integrated Discovery (DAVID) [43]. Annotated ESTs were categorized based on the terms in each of the standard GO categories, specifi- cally biological process (BP), molecular function (MF) and cellular component (CC). Authors' contributions MBS, TLC and MJM-F are the Principal Investigators who were involved in the design of this study. MBS designed and coordinated all aspects of the project. MJM-F con- ceived the study, coordinated tissue collection and obtained funding for research reported in this manuscript. TLC coordinated the creation of the EST databases. MFB coordinated the creation of the cDNA libraries and the EST databases. CKC, JVT, MSG, WJC-G, AC, TD, BJ, TAK, CY and JWR collected tissue for the creation of these libraries. JJS and JLK participated in isolating RNA for the creation of these libraries. In addition, CKC participated in the design and annotation of the EST database, per- formed gene ontology analyses and drafted the manu- script. ME and SW created the cDNA libraries. KC, TK and Page 8 of 10 (page number not for citation purposes)