BMC Genomics 2006, 7:154 Table 1: EST library descriptions. Library Number of ESTs UI-S-GB0 UI-S-GB1 UI-S-GG0 UI-S-GG1 UI-S-GN0 UI-S-GN1 UI-S-GS0 UI-S-GS1 UI-S-GU0 UI-S-GU1 UI-S-HH0 1,174 4,481 964 4,292 1,552 5,187 1,839 4,151 4,472 2,079 5,171 Description Aposymbiotic; 0 hours Aposymbiotic; 0 hours; normalized Aposymbiotic; 12 hours Aposymbiotic; 12 hours; normalized Symbiotic; 12 hours Symbiotic; 12 hours; normalized Aposymbiotic; 48 hours Aposymbiotic; 48 hours; normalized Symbiotic; 48 hours Symbiotic; 48 hours; normalized Pooled, subtracted library from UI-S-GB1, -GG1, -GN1, -GS1, -GU1 http://www.biomedcentral.com/1471-2164/7/154 ies, 1,381 from 12-h aposymbiotic libraries and 1,314 from 48-h aposymbiotic libraries) and 34% (4,738) are derived from only symbiotic libraries (1,699 from 12-h symbiotic libraries and 3,039 from 48-h symbiotic librar- ies). Cluster analysis also revealed that 55% (7,639) of 13,962 clusters are singleton clusters (representative of only one FNiogvuerlege3ne discovery Novel gene discovery. Large increases in sequence contri- butions are evident from each set of libraries, which are illus- trated as follows: sequences from 5 non-normalized libraries (approximately the first 10,000 ESTs); sequences from 5 nor- malized libraries (approximately from 10,000 to 30,000 ESTs); and sequences from pooled, subtracted libraries (after 30,000 ESTs) (Table 1). Novelty rates, calculated as the ratio of the total number of clusters identified over the total number of ESTs generated, were 39%, 54%, and 75%, for the non-normalized, normalized and subtracted libraries, after production of a total of 10,000 (3,920 clusters), 20,190 (10,822 clusters), and 5,171 (3,917 clusters) ESTs, respec- tively. EST) and of these, 37% (2,794) are associated with a known protein, 23% (1,720) with a hypothetical protein, and 7% (523) with an unknown protein at an E-value threshold of e-03. Of the singleton clusters, 16% (1,221) do not have a BLAST hit and may be novel. In the set of clusters containing more than 1 EST, 52% (3,271) are associated with a known protein, 17% (1,073) hit with a hypothetical protein, and 6% (351) match with an unknown protein at an E-value threshold of e-03. Of the remaining 45% (6,323) of clusters containing two or more ESTs, 12% (742) do not have a BLAST hit. Although most of these clusters are represented by two or three ESTs, more than a quarter are composed of at least four ESTs with one cluster of 57 ESTs. The similarity of E. scolopes sequences to those of three animals that are commonly studied but distantly related to E. scolopes, Homo sapiens, Drosophila melanogaster and Caenorhabditis elegans, were determined using a BLAST- based analysis with an E-value threshold of e-50. The goal of this analysis was to perform a comparative genomic analysis through identification of orthologous genes. This is an alternate strategy which is based on gene content as opposed to a traditional molecular phylogeny analysis which is based on gene sequence. We evaluated several genes using the PHYLIP package (data not shown) with indeterminate results[22]. The results of these analyses indicate the squid ESTs have the greatest number of genes with similarity only to H. sapiens genes with 1.30% (342) of the 26,309 annotated human sequences, compared to 0.22% (40) of the 18,289 annotated sequences reported in D. melanogaster and 0.03% (6) of the 22,215 annotated sequences reported in C. elegans (see Additional file 4). These data show a higher number of squid genes homol- ogous to a deuterostome chordate, H. sapiens, than to two members of the Ecdysozoa, the arthropod D. melanogaster and the nematode C. elegans. To assess characteristics of the untranslated region and codon usage, a BLAST search was performed between the Page 5 of 10 (page number not for citation purposes)