BMC Genomics 2006, 7:154 http://www.biomedcentral.com/1471-2164/7/154 FThigeulrigeht1organ system of E. scolopes The light organ system of E. scolopes. A. A swimming adult animal. The light organ (internal to the boxed area) is located in the center of the mantle cavity. Bar, 1 cm. B. A confocal image of a light organ (labeled with CellTracker, Molecular Probes) within a diagram of a newly hatched ani- mal. The juvenile organ has a complex, superficial ciliated epi- thelium (sce) that facilitates colonization by the symbioint. Bar, 200 microns. C. A confocal image of the sites of V. fischeri entry into host tissues (labeled with acridine organ, AO). A set of three pores, into which aggregated V. fischeri cells will migrate, are located at the base of each lateral field of ciliated epithelial cells. In response to interactions with colonizing V. fischeri, host hemocytes (h) migrate into the sce, which will be lost during symbiont-induced light organ mor- phogenesis. Also visible in this image is the condensed chro- matin characteristic of symbiont-induced apoptosis (arrows), which stains vividly with AO. Bar, 20 microns. D. A histologi- cal section revealing the path traversed by colonizing symbi- onts. Once aggregated in mucus outside the light organ, the symbionts enter the pores (p), travel up long ciliated ducts (d) and enter the crypt spaces where they interact with two cell types, the polarized epithelium that lines the crypts (ce) and a transient population of host hemocytes (h). Bar, 30 microns. ated 11 cDNA libraries derived from expressed sequences tags (ESTs) isolated from three developmentally signifi- cant time points in the squid-vibrio system. This approach has been successfully used in the past to identify the set of transcribed genes specific to an organ or tissue of a partic- ular organism (see e.g., [16-20]). The generation of these resources paves the way for an in-depth examination of the influence of symbiosis in host gene expression in the squid-vibrio system. Results A total of 11 cDNA libraries (Table 1) were generated from 5 pools of squid light organ RNA, containing clones with insert sizes ranging from 0.35 to 2.5 kb and an average length of ~1 kb. From these cDNA libraries, sequencing generated a total of 42,564 3' EST sequences with an aver- age sequence length of 692 bp and an average phred value of 39.7. An overall novelty of greater than 45% remained throughout the sequencing phases of the non-normal- ized, normalized and subtracted libraries (Fig. 3). Of the 42,564 ESTs generated, 35,421 ESTs satisfied the quality criteria (see Materials and Methods). From the sequences that passed the quality criteria 97% (34,249) contained a polyadenylation tail. Of those sequences with a polyadenylation tail, 63% (21,439) contained a canon- ical polyadenylation signal sequence (AATAAA or ATTAAA), and another 15% (5,157) contained an alterna- tive polyadenylation signal sequence[21]. In silico com- parison of these 42,564 ESTs to sequences from the V. fischeri genome showed no sequence similarity, suggest- ing neither contaminating V. fischeri sequence nor evi- dence for horizontal gene transfer. These 35,421 ESTs were divided into 13,962 unique clus- ters using the UIcluster program. One sequence from each cluster was chosen to be a part of a nonredundant set of ESTs. Within this set of clusters, 13,441 contained at least one sequence with a polyadenylation tail and 8,806 of these also contained a polyadenylation signal sequence. Five prime sequences with an average length of 683 bp were generated from these nonredundant representative sequences from each cluster. Of these sequences, 58% (8,067) of 13,962 ESTs contained sufficient overlapping paired 3' and 5' sequences to form contigs with an average sequence length of 1,065 bp. These contigs were used in place of the individual 3' and 5' sequences in further anal- yses when available. Representative 3' and 5' sequences from the final non-redundant set of ESTs were deposited in the dbEST division of GenBank. The BLAST annotation of the nonredundant set (13,962 ESTs) showed that 70% (9,728) had a significant hit to a protein in the NCBI nonredundant protein database with an E-value threshold of e-03 (Fig. 4). Of these significant hits, 62% (6,061) hit an annotated known protein, 29% (2,793) hit a hypothetical protein and 9% (874) hit an Page 3 of 10 (page number not for citation purposes)