BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 Results: Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion: Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data. Background Genome sequence determination of the model organism Drosophila melanogaster was a landmark that launched a new era for functional genomic studies in complex organ- isms. The almost complete version of the euchromatic DNA sequence was first released in March 2000 due to a collaborative effort of the Drosophila Genome Projects and Celera Genomics [1]. Using gene prediction softwares in combination with searches of protein and EST databases, initial in silico analyses indicated the existence of 13,601 protein-coding genes (PCG), an extraordinarily small number of genes when compared to the approximately 19.000 PCG encoded in the C.elegans genome [1]. After the release 1, an intensive collective work took place in order to improve sequence quality and annotation, fill in the gaps, and correct the assembly. With the aim of gen- erating the information necessary to define the transcripts encoded in the genome, the Berkeley Drosophila Genome Project (BDGP) initiated a high throughput production of both EST and full length cDNA sequences based on con- ventional and normalized cDNA libraries from different tissues and developmental stages [2]. This effort was fol- lowed by non-BDGP projects with a major contribution from the Exelixis Drosophila melanogaster EST project, which has adopted sequencing of random primed librar- ies of mixed stage embryos, imaginal disks, and adult heads to increase the transcription units coverage [3]. Cur- rently, there are about 39,346 full length mRNA and 532,557 EST sequences available in the NCBI database, totalizing approximately 16,681 clusters according to UniGene [4]. Since the year 2000, several subsequent genome versions have been released, each one improved by BDGP and annotated by FlyBase [5]. Release 3.2, con- sidered the first finished version, was published in March 2004 and provided a complete revision of all gene models and other genome features [6], estimating a total number of 13,792 PCGs plus 527 non-protein coding genes (tRNAs, rRNAs, microRNAs, sn/snoRNAs). Release 4.3, the last annotated genome version published in March 2006, includes a total of 14,816 genes and is available for searches by gene annotation, BLAST or sequence ID at the FlyBase website [5]. During the last few years, an enthusiastic debate about the number of PCGs in the organisms with sequenced genomes has arisen. For D. melanogaster, estimates varied from the initial ~ 13,600 coding gene predictions [7] to about 16,000 gene predictions, based on microarray expression data [8]. A careful computational and experi- mental analysis carried to validate the Drosophila genome annotation has recently concluded that the D.melanogaster genome in fact contains approximately 14,000 protein- coding genes, although some genes presenting unusual features that make them refractory to prediction methods may remain to be discovered [9]. However, the truthful notion about the complexity of the D. melanogaster tran- scriptome is still under construction. In this respect, it has been inferred from DNA oligonucleotide microarrays, with unique sequences tiled throughout the genome and across predicted splice junctions, that over 40% of the Drosophila genes contain one or more alternative exons [10]. Additionally, a transcription map with a 35 bp reso- lution of the initial 24 hours of development indicates that 30% of the transcribed regions are still unannotated. Approximately 23% of these are intronic and 7% corre- spond to intergenic regions. Based on manual and com- putational surveys designed to identify coregulated expression patterns between unannotated and annotated genome regions, it was estimated that 29% of the unanno- tated regions are part of transcripts incompletely anno- tated or potential alternative exons of known genes [11]. Therefore, correcting and refining the genome annotation is a reiterative task, which is continuously being done and depends on experimental data for final validation, espe- cially for the identification of rare transcripts and alterna- Page 2 of 9 (page number not for citation purposes)