BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 aSFtiag2tu3isrtDeicAa1lPly, ecnormicphaerdedGweniteh Olenvteol l2ogGyOtertemrsmlsevfoelr2alaltopth≤er0.A0.5thinalBia.nnaapgeusneseleodcsi for LongSAGE tag-matched A. thaliana gene loci Statistically enriched Gene Ontology terms level 2 at p ≤ 0.05 in B. napus seeds for LongSAGE tag-matched A. thaliana gene loci at 23 DAP, compared with level 2 GO terms for all other A. thaliana gene loci. The plot was produced using the online tool WEGO (Ye et al., 2006). development, water and fluid transport, cell-cell signal- ling, cell wall modification and glycerolipid, neutral lipid, acylglycerol, triglyceride, and carbohydrate metabolic processes at 23 DAP, and in protein processing, protein targeting, photosynthesis, fatty acid biosynthesis, lipid localization, storage and lipid metabolic processes at 35 DAP. Genes matched in sense and antisense orientation In the 23 DAP library, 6 of the 30 most abundant tags matched in sense as well as in antisense orientation to the Brassica EST dataset, oriented based on the A. thaliana pro- teome. The same was true for 10 of the 30 mosXt abun- dant tags at 35 DAP. In most of these cases 500 to 1,500 ESTs were matched by a single tag in one direction, whereas only 1 to 13 ESTs were matched by the same tag in the opposite direction. A detailed analysis of these matches revealed that in all cases the smaller fraction of matched ESTs were chimeric sequences composed of two regions, derived from different gene loci, that aligned to the A. thaliana proteome or to B. napus full-length cDNAs in different orientations. These apparently chimeric ESTs may represent cloning artefacts derived from tail-to-tail or head-to-head ligations during cDNA preparation for EST library production. In some cases a putative chimeric composition was also found for individual ESTs, with two different regions derived from different genes being aligned in sense and antisense orientation to the A. thal- iana proteome or to oriented B. napus full-length cDNAs. To remove these putative chimeric ESTs from our analysis, tag-to-EST-to-A. thaliana gene locus matches were only accepted if at least 4 tag-matched Brassica ESTs matching a particular A. thaliana locus were found. In addition, tag- to-EST-to-gene matches of particular tags matching in sense as well as in antisense orientation were only included in the analysis if the frequency of ESTs matched in one direction was at least 1% of the frequency of ESTs matched in the other direction. After removal of putative chimeric ESTs, the number of accepted tags that were suc- cessfully matched via Brassica ESTs to A. thaliana genes was reduced strongly from 8,255 to 5,120 (Table 2), due to the exclusion of many low-copy ESTs from the analysis. The occurrence of chimeric ESTs in databases has been documented previously. For example, Hillier et al. [21] found chimeric ESTs in a dataset of 280,000 human ESTs at a frequency of up to 1.04%. The strategy we applied Page 9 of 16 (page number not for citation purposes)