BMC Genomics 2007, 8:42 http://www.biomedcentral.com/1471-2164/8/42 (nucleotide position 18–290 from the TSS in isoform a, and position 164–400 in isoform b), CNGA3 (position 166–315), DHRS3 (position 166–315), KIFC3 (position 4–195), and SLC24A2 (position 55–183 isoform a, 44– 289 isoform b). Yet the translated sequences of these short ORFs do not have homology with any protein in public databases. Detection of novel splicing variants and shorter transcripts Our experimental procedure described alternatively spliced isoforms for two genes IMPG1, SAG, which lack exon 2 of the RefSeq. These forms have not been anno- tated in the RefSeq database. We confirmed these alterna- tively spliced isoforms by regular RT-PCR (Data not shown). The second exon of the gene SAG contains the TSS and the presence of this alternative form, lacking the regular start site, may play a role in the regulation and fur- ther processing of the transcript. For 4 genes (CRB2, CRX, RP1, WDR17), we detected shorter transcripts that lack the annotated start codon. Since these experiments were done with the same adapter ligated first-strand cDNA we assume that these short transcripts are derived from true alternative TSSs. These transcripts may be preferentially amplified in the RT-PCR and may be translated from an internal initiation codon. We report in Table 1 the detailed results for these genes. Confirmation of results with primer extension To provide an experimental validation of our results we undertook primer extension experiments. We performed reverse transcription of mRNA with a sequence-specific FAM-labelled primer for two genes (CNGA3, RDH12). The length of the FAM-labelled cDNA primer extension product can be analysed on ABI-DNA Genetic Analyser using GeneScan software. As a result of the analyses we detected a fragment of 350 bp for CNGA3 (Fig 2A) and a fragment of 215 bp for RDH12 (Figure 2B). The size of these fragments confirms the presence of the transcripts that we detected with RACE. Comparison with existing annotations and databases To assess the quality of current annotations of the 5' end of genes expressed in human retina, the sequences obtained by 5' RACE were compared with the correspond- ing gene annotation/prediction. Overall, RACE experi- ments detected 15 exons that were neither annotated nor predicted for retina transcripts; 8 exons did not have any matching experimental evidence in GenBank, while the other 7 showed different boundaries or alternative splice sites. Of these 15 un-annotated exons, 12 are first exons and can be considered the new first exon for the retinal transcripts. Of the 54 genes successfully amplified, 41 (76%) delivered 5' RACE sequences different from the annotation. Results of a parallel project, DBTSS [8], sup- ported our results concerning 3 of these genes (CNGA3, ELOVL5 and SLC24A2) although the source of mRNA was not human retina. We extended the annotated first exon of 27 RefSeq genes by an average of 60 transcribed bases. We compared our results with genome wide mapping of TSSs using CAGE tags [10]. We found perfect correspond- ence for 13 transcript isoforms; for another 6 transcripts the start site retrieved in the CAGE database is located less than 400 bp away. For 35 transcript isoforms the TSS is located in a different position (See Table 1). This discrep- ancy in the results may be due to the fact that the CAGE database doesn't include retina amongst the panel of ana- lysed tissues and therefore lack specific and rare transcript isoforms present in that tissue. Shape of TSSs and conservation After analyzing the distribution of RACE clones we could define the shape of TSSs according to the classification previously reported [10]. The different clones were clus- tered and depending on the start base position of each clone within a cluster we divided the start sites into four shapes. In the single dominant peak class (SP) the major- ity of clones are concentrated to no more than four con- secutive start positions with a single dominant TSS. The clusters spanning a broader region are grouped in a gen- eral broad distribution (BR), a broad distribution with a dominant peak (PB) and a bi- or multimodal distribution (MU): 22 genes showed a single dominant peak, 11 a broad distribution, 8 a bi-multi peak distribution and 6 a broad distribution with a dominant peak. For some tran- scripts we could not make a classification because the number of clones was less than 5. We report the results of this analysis Table 1 (TSS shape). Figure 3 shows a graph- ical view of TSSs identified for AOC2, ABCA4, RDH12, and LRRC21 as an example of the different distributions observed. The classification of the shape of TSSs defined by distribution of 5' end RACE clones within a cluster is useful for the further characterization of expression regu- lation. The distribution of the clones defines different ele- ments of the core promoter and gives insights on the start of transcription. Even if broad promoters are the major class in mammals [10], 36 of the analysed transcripts present a dominant peak highlighting the possibility that those transcripts are tightly regulated. Although TSSs of orthologous genes do not necessary reside on equivalent locations because of evolution of mammalian TSSs [30], we analysed sequence conserva- tion of the first new exons among a set of mammals (mouse, dog, cow): the range of conservation varies between 42 and 89 %. We report the pairwise alignment percentage of identity in Additional file 7. Sequences residing upstream and downstream from the boundaries of new defined exons are regions displaying high regulatory potential calculated by a computational Page 7 of 14 (page number not for citation purposes)