BMC Genomics 2006, 7:154 http://www.biomedcentral.com/1471-2164/7/154 sFAeinqgnuuoertneact4eiosn of 13,962 unique E. scolopes light organ Annotation of 13,962 unique E. scolopes light organ sequences. A. A pie chart indicating the number of genes and the percent of ESTs annotated as a known protein, unknown protein or a hypothetical protein. All sequences that are annotated as a known protein or as significant (sig) have a BLASTX hit below the E-value threshold of e-03. B. A histogram illustrating the total number of unique E. scolopes ESTs, the total number of ESTs annotated as a known pro- tein with an E-value threshold of e-03, the total number of ESTs annotated with at least one category of Gene Ontology (GO) and the number of genes annotated in each of the 3 major GO categories, biological process, molecular function and cellular component. SwissProt amino acid databases and the assembled con- tigs. A total of 8% (627) of the 8,067 assembled contigs contained a BLAST hit of at least e-40 with a stop consist- ent with the carboxy terminus of at least one protein (+/- 5 AA). This same analysis estimated an average 3' UTR length of 227 bp. This estimate is conservative as genes with a UTR longer than the average contig length of 1,065 bp may not have a BLAST hit. These same UTR predictions were used to estimate the GC content in the coding sequence compared to the untranslated sequence. A bias in GC content was observed between the coding and non- coding sequence from 31.5% GC in the predicted untrans- lated sequence to 42.4% GC content in the predicted cod- ing sequence. Of the nine least frequently used codons six are exclusively composed of G's and C's. These codons were significantly under-used ranging in prevalence from 0.5% – 0.75%. However, the codon GGC, occurs at a fre- quency of 1.3%, and the codon GCC, occurs at a fre- quency of 1.6%, and due to the increased prevalence, these two codons are considered significant outliers. Discussion In this report, we characterize 13,962 nonredundant EST sequences generated from eleven cDNA libraries derived from pools of juvenile E. scolopes light organs, which were collected at developmentally significant time points with and without colonization by V. fischeri. These nonredun- dant sequences were: 1) characterized, 2) annotated, and 3) analyzed for novelty within each condition. UniGene sets of over 40,000 unique transcripts have been reported for vertebrate chordates. However, among the invertebrate chordates and other invertebrates, they are most often less than half that size, ranging from approxi- mately 6,000 to 15,000 unique transcripts. Based on clus- tering analysis, which estimates the number of transcripts present in the organism, the E. scolopes nonredundant EST set containing 13,962 clusters is in the same range as that of other invertebrates represented in the UniGene data- base. It is interesting to note, that the UniGene database reports unique sets as composites of all reported EST sequences, i.e., it is not organ specific, thus, the E. scolopes nonredundant set is relatively large for an organ specific library and may indicate a substantially larger transcrip- tome than has previously been reported for other inverte- brates. A comparison of the EST clusters derived from the eleven squid light organ libraries showed that the largest number of unique clusters is associated with the 48-h symbiotic condition. This increase in transcriptional diversity is supported by two-dimensional polyacryla- mide gel electrophoresis analysis of standing stock protein in the developing squid light organ, which showed that differences in light organ protein profiles in response to V. fischeri colonization are not easily detectable until 48 hours post-inoculation [23]. Sequencing of the eleven squid light organ cDNA libraries resulted in ESTs of similar length and quality to sequences generated from previously constructed cDNA libraries [16-18,24]. The predicted 3'-UTR length of 227 bp is shorter than the 411 bp 3'-UTR length found for ortholo- gous human and rat genes [25]. This may be explained, at least in part, by the occurrence of A-rich sequences within the 3' UTRs of a significant fraction of E. scolopes tran- scripts. This hypothesis is based on the fact that 22% of the squid ESTs do not contain a recognizable polyade- nylation signal sequence within 30 bases from the 3' end Page 6 of 10 (page number not for citation purposes)