BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 tive splice variants. With the aim of covering the diversity of transcripts expressed in Drosophila the generation of EST information from different sources is currently under way [2,3]. Here we use the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is based on low stringency RT-PCR, to generate D. melanogaster expressed sequence information. ORESTES are preferentially derived from the central coding portions of the transcript and frequently identify less abundant messages [12,13]. Such approach was previously applied for human tran- scriptome characterization, validating a large percentage of genes and identifying 219 unannotated transcribed sequences on chromosome 22 [14]. More recently, a large-scale analysis of ORESTES derived from head, neck and thyroid tumors pointed to 788 putative new alterna- tive splicing isoforms. A subset of 34 was submitted to experimental validation resulting in the confirmation of 23 (68%) new alternate exons [15]. Analysis of 1,303 Drosophila ORESTES clusters revealed 68 potential transcribed regions unannotated in the current version of the genome (release 4.3). Experimental valida- tion of 38 (~ 50%) of this unannotated ORESTES revealed 17 new exons that most likely belong to low abundance transcripts. Using the ORESTES information together with a PCR based approach we obtained the complete coding sequence of a new serine protease which mRNA expres- sion is induced upon infection. Our data reinforce the importance of PCR based methodologies for refining the Drosophila transcriptome, particularly for the identifica- tion of previously unannotated low copy transcripts. Results and discussion ORESTES in silico analysis Of 1,303 Drosophila ORESTES clusters from different developmental stages of D.melanogaster, 176 were identi- fied as unannotated in the genome release 1 (see material and methods). We re-analysed this set of 176 ORESTES against the current annotated version of the genome (Fig- ure 1). Sixty-eight of these clusters aligned in unannotated genome regions indicating putative new exons (GeneBank_Accn. EG974084 to EG974151). Fifty-three out of the 68 clusters either match or overlap ESTs gener- ated by other projects [2,3], suggesting that they indeed represent true transcripts. Amongst the remaining clusters, 98 overlap annotated exons represented by cloned cDNAs and ESTs, 8 correspond to transposons or repetitive sequences and 2 are chimeric. About half of the 68 unan- notated ORESTES (38) were selected for validation by developmental northern blot hybridization. sFAoinpgahuliylraseism1felolawnocghaasrtterofge1n7o6mOeRvEeSrTsiEoSnu1nannotated in Dro- Analysis flowchart of 176 ORESTES unannotated in Drosophila melanogaster genome version 1. Analysis against the genome assembly 4.3 and expressed sequences were manually performed using BLASTn at the FlyBase [5] and BLAT Search [16]. 50% of the unannotated exons detected by ORESTES belong to low abundance transcripts Of the 38 ORESTES that mapped in unannotated exons of the version 4.3 of the genome, 17 (50%) detected mRNAs only in polyA+ northern blots (Figure 2) indicating that they all represent low abundance mRNAs. The other 21 ESTs could also represent transcribed regions that cannot be detected by our analysis, such as precursors of small non-coding RNAs [17]. Among the 17 validated Page 3 of 9 (page number not for citation purposes)