BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 resulted in removal of all 72 chimeric ESTs that were iden- tified by manual analysis of the 47,446 ESTs matched by the 30 most abundant tags in the two LongSAGE libraries. Based on the tag-to-gene matching strategy described above it was found that 24.3% of all matched tags matched A. thaliana genes via oriented Brassica ESTs in antisense orientation (corresponding to 1762 of 5,120, 34.4% of all matched genes, Table 2). In the 23 and 35 DAP libraries, only 68.3% and 74.6% of the respective genes were matched exclusively by sense tags (Table 2). On the other hand, 6.6% and 6.8% of all genes were matched exclusively by antisense tags at 23 DAP and 35 DAP, respectively, while 25.1% (23 DAP) and 18.6% (35 DAP) of the genes were matched by both sense and anti- sense tags. Genes expressed with high abundance in sense orientation, and particularly members of complex gene families, often also showed coexpression of antisense transcripts at a lower frequency (Figure 2, 275 differen- tially expressed genes matched in sense and antisense ori- entation). A detailed analysis of tags matching to the four 2S seed storage proteins of A. thaliana (AT4G27140, AT4G27150, AT4G27160, AT4G27170) was performed by aligning them to Brassica spp. napin cDNAs and genomic napin sequences. This revealed that some tags matched in antisense orientation to highly conserved regions of napin transcripts, derived from multiple B. napus loci, whereas other tags matched in sense orienta- tion to highly diverse regions of napin transcripts derived from a limited number of B. napus loci. Detection of napin-related sense and antisense transcript expression by Real-time RT-PCR At 35 DAP, a high number of tags matched in sense orien- tation to Brassica ESTs that were similar to A. thaliana pro- teins containing the AAI_SS domain (NCBI Conserved Domain Database cd00261). This protein domain is found in the alpha-amylase inhibitors (AAIs) and seed storage (SS) protein subfamily including plant lipid trans- fer proteins, seed storage proteins and trypsin-alpha amy- lase inhibitors. At 35 DAP the most abundantly expressed tags were matched via Brassica ESTs in sense orientation to the four A. thaliana 2S seed storage proteins 1–4 (napins) AT4G27140, AT4G27250, AT4G27160 and AT4G27170 sFHeiengasutermeana2dp/orf naonrtimseanliszeedorLioentgaStAioGnEatco2u3nDtsAfoPra3n7d53d5ifDfeArePntially expressed A. thaliana genes matched by LongSAGE tags in Heat map of normalized LongSAGE counts for 375 differentially expressed A. thaliana genes matched by Long- SAGE tags in sense and/or antisense orientation at 23 DAP and 35 DAP. Expression is represented on a logarithmic scale. The descriptions of the 10 most abundantly expressed genes in the respective libraries are highlighted in red for 23 DAP, in blue for 35 DAP or in black for genes that were among the 10 most abundant at both time-points. If no expression was detected counts were set to 0.1 (black squares). Page 10 of 16 (page number not for citation purposes)