BMC Genomics 2007, 8:249 http://www.biomedcentral.com/1471-2164/8/249 ORESTES, 4 are derived from the embryo library (DE), 3 are from the library constructed with RNA extracted from larvae, prepupae plus pupae (DL), 5 are from an adult library (DA) and 5 are from a library constructed with a mixture of the RNA from all developmental stages (DP). Based on these data and in the number of ORESTES clus- ters produced from the DE (113), DL (430), DA (887) and DP (729) libraries, the estimated discovery rate of new exons for each library was 35.4, 7.0, 5.6 and 6.9 new exons per 1,000 ORESTES from the DE, DL, DA and DP libraries, respectively. The 5–6 higher discovery rate found for DE library indicates that, despite the large amount of embryo ESTs available in the public database, the embry- onic transcriptome needs further sequencing for its defini- tion, which is in accordance with previous transcriptional analysis of the Drosophila embryogenesis performed at the genomic scale [11]. The majority of the validated ORESTES (14) detected a single band in northen blots, which could indicate the presence of unique transcripts. Three ORESTES detected more than one mRNA species (Figure 2, ORE-3, ORE-15, ORE-16), which could either constitute isoforms of the same gene or mRNAs encoded by different genes sharing common exons. Ten of these 17 validated ORESTES detected transcripts in all analysed developmental stages, namely: embryo, larvae and adult (Figure 2; ORE-1, -2, -3, -5, -6, -9, -11, -12, -14, -15). The other seven (Figure 2; ORE -4, -7, -8, -10, -13, -16, -17) detected stage specific transcripts. ORE-4 and ORE-10 detect mRNAs of 4.5 and 11.5 kb, respectively, which are mainly expressed in embryos. The 8.5 kb mRNA detected by ORE-17 is abun- dant in embryos and present in lower amounts in adults. ORE-12 detects an 11.0 kb transcript present mainly in embryo and adults. The transcripts of about 0.9 kb and 3.5 kb detected by ORE-8 and ORE-13, respectively, were only detected in larvae and adults. ORE-7 detects a 0.6 kb mRNA present at higher levels in larvae that is also expressed in embryos. ORE-3 detected one mRNA of about 4.5 kb exclusively expressed in embryos, and a 2.1 kb mRNA present at all stages. ORE-15 detected three mRNAs of 9.8, 5.0 and 1.8 kb. The 9.8 kb mRNA is mainly detected in larvae; the 5.0 kb RNA is much more abundant in embryos, but also detected in adults; and the 1.8 kb RNA is exclusively expressed in adults. ORE-16 detected 3 different transcripts of about 4.0, 3.1 and 2.2 kb, all of them observed in adult flies. Altogether, our ORESTES analyses resulted in the valida- tion of 17 novel exons. Of these, five ESTs constitute, up to now, the unique expressed sequence information for the corresponding genomic regions (Table 1), despite the wealth of cDNA data available for Drosophila, and the ongoing projects of cDNA/EST generation using tissue specific and normalized cDNA libraries [3,18,19]. Both gnFDeoiegntvaouetmrleoedep2imnetnhtealcpurorefinlet voef rtsriaonnsc(r4i.p3t)socfotnhteaiDni.nmgeelaxnoongsasutenran- Developmental profile of transcripts containing exons unannotated in the current version (4.3) of the D.melanogaster genome. Autoradiograms of Northern blots containing poly A+ RNA extracted from embryos (E), third instar larvae (L3), and adults (A), which were hybridized to the probes indicated at the bottom of each blot. The esti- mated size (kb) of each transcript is indicated on the left. Page 4 of 9 (page number not for citation purposes)