BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 sequences were also obtained using different napin tag primers, with up to 30% divergence, within 200 bp of the 3'-UTR, from 16 clones. These observations suggest that a high number of distinct protease inhibitor/seed storage/ lipid transfer protein (LTP) family protein genes or tran- script variants are expressed in B. napus seeds. Also these data support the increasing recognition of the high com- plexity and variability of 3' UTR regions in genes from higher organisms [13,15,16]. Based on the cDNA library production using polyA+ trapped mRNA, LongSAGE tags are expected to match at the anchoring enzyme site closest to the 3' end of full- length ESTs expressed in sense orientation (canonical position). However, only 95.3% of the matched tags in the sense EST dataset were matched in the last canonical position. There are numerous possible explanations for the high frequency of tags matching a non-canonical posi- tion. In particular, they could arise from (a) cloning arte- facts caused by partial digestion with the anchoring enzyme NlaIII during SAGE library preparation, (b) alter- native transcript variants with varying length derived from one gene locus, or (c) EST cloning artefacts creating chi- meric EST molecules derived from two or more different genes. Also, due to partial EST sequences present in the dataset, a non-canonical position might be falsely anno- tated to the canonical position. Except in the third case, non-canonical tags are still valid tags for accurate annota- tion. We attempted to reduce annotation artefacts (c) by removing putative chimeric ESTs from analysis (see sec- tion 'Genes matched in sense and antisense orientation' for details). This increased the ratio of tags matched to the canonical position of oriented Brassica ESTs from 95.3% to 98.3%. However, even after filtering out putative exper- imental artefacts using this approach, only 77% of all 243,528 ESTs matched by tags in the canonical position are matched exclusively at this position. Instead, 23% of these individual ESTs are additionally matched by tags at other non-canonical positions (up to the 9th before last position), suggesting that the usage of alternative tran- scripts (e.g. by alternative polyadenylation) is a common mechanism. In other words, a high diversity exits in the 3' UTR of many expressed transcripts, as has been described before in humans [13]. No information exists about tran- scription start points for polyA+ containing transcripts expressed in antisense orientation. Therefore different proportions of matched antisense tags might be expected at canonical and non-canonical positions. In contrast to the tags matched in sense orientation, only 65.5% of tags were matched to canonical positions for the tags matched in antisense orientation. Comparison of the most abundantly expressed tags and assigned genes Tag-to-EST matches based on 15,955 tags were assigned to a total of 8,255 A. thaliana gene loci at both time-points (Table 2). About 85% of the tags (13,641) were assigned exclusively to a single A. thaliana gene locus. The other 15% of the tags matched 2 to 45 loci, with some tags matching more than 10 genes exhibiting a polyA-like homopolymer composition. Due to the positioning of the anchoring enzyme restriction site close to the polyA tail in some transcripts, these contained stretches with up to 16 adenosine bases derived from phylogenetically unrelated genes. Other tags matching more than 2 genes were clearly derived from groups of phylogenetically related genes. For example, the tag CATGAACAGTTTCATCAACGA matched to six different members of the histone gene family (AT2G37470, AT3G53650, AT5G22880, AT5G59910, AT1G07790 and AT5G02570). On the other hand, some A. thaliana loci were matched by up to 96 different B. napus tags via the Brassica ESTs. More- over, some loci were not only matched by different tags, but these tags also matched at different positions within one particular matched EST molecule. These multiple matches underline the complex paleopolyploid structure of the B. napus genome in comparison to A. thaliana [17]. For example, the 4 A. thaliana gene loci of the 2S seed stor- age protein family were matched by 47 different tags, including 9 of the 30 most abundant tags, at 35 DAP. Due to this complexity, a three-step procedure was applied for calculating average values of the relative abundance of gene expression, based on the tag-to-gene matching results for each single A. thaliana gene locus. In a first step, the measured counts were evenly distributed to the matched gene loci if a tag matched more than one gene locus. In a second step, tag counts were added together if different tags matched the same gene locus. In a final step the summed tag counts for each gene locus were normal- ized to a total tag count of 1,000,000 for both libraries and the relative abundances were calculated. Average cumulative counts and frequencies for the 20 most abundantly expressed genes at 23 and 35 DAP are shown in Table 4 and 5, respectively. The most abundant transcripts at 23 DAP, with 1.59% of all matched tag counts, correspond to the A. thaliana senescence-associ- ated cysteine-type protease SAG12 (AT5G45890), which was also among the 20 most highly-expressed genes at 35 DAP. Two protease inhibitor/seed storage/lipid transfer family proteins (AT5G38195, AT1G48750) were also among the 20 most highly expressed genes at both time- points. Other highly expressed transcripts at 23 DAP are a number of catalytic enzymes and other genes that are involved in diverse biological processes. At 35 DAP, tran- scripts related to the four closely-related A. thaliana 2S seed storage protein genes showed the highest counts, with a cumulative abundance of about 10%. Other highly expressed transcripts at 35 DAP are related to other storage proteins (cruciferin, oleosin), protease inhibitor or lipid transfer proteins and genes involved in fatty acid biosyn- Page 5 of 16 (page number not for citation purposes)