BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 ray analyses (e.g., [3,4]). These studies revealed major gene expression changes during seed filling and desicca- tion, along with distinct expression patterns related to car- bohydrate metabolism, lipid biosynthesis and storage protein accumulation. However, the regulatory mecha- nisms that ensure the proper execution of seed develop- ment in A. thaliana and other plants remain largely unknown. Furthermore, generalisation of findings regard- ing gene regulation from model to crop plants is difficult because most major crops have considerably more com- plex genomes. Oilseed rape (Brassica napus ssp. napus), the closest major crop relative of A. thaliana, is the world's second most important oilseed crop. The high-value nutritional oil is also an excellent substrate for biodiesel production, whereas the protein-rich seed meal remaining after oil extraction is a valuable livestock feed. Oligonucleotide microarrays constructed for A. thaliana have been used in the past for expression profiling in B. napus, but have not provided optimal signal intensity and reproducibility [4- 7]. Recently the total number of ESTs from Brassica species deposited in public databases has risen dramatically to more than 800,000 entries with about 280,000 from seed developmental stages. 67,000 ESTs from seed develop- mental stages have been used to develop a B. napus cDNA microarray for analysis of seed gene expression patterns [7]. An alternative for accurate, quantitative global expression profiling is serial analysis of gene expression (SAGE; [8]), which in contrast to microarray hybridization allows the detection of new transcripts. SAGE is an expression profil- ing technique that simultaneously measures the levels of thousands of genes expressed in a given tissue. The method is based on the excision of short tags from poly A+ RNAs and end-to-end ligation of ditags to form high molecular weight concatemers. This allows cost-effective high-throughput cloning and sequencing of concatemers. Matching of tags to genomic sequences is a critical step in SAGE data analysis, and normally this requires the availa- bility of high quality genome annotation data [9]. SAGE was first used for quantification of gene expression in human using 13–15 bp tags [8]. Modifications of the orig- inal SAGE protocols producing 21 bp tags (LongSAGE; [9]) and 26 bp tags (SuperSAGE; [10]) have been devel- oped to enable more efficient and unambiguous tag-to- gene assignment in higher organisms with more complex transcriptomes. SAGE is commonly used in animal genomics, but has been increasingly used in plant species and tissues [11]. The intention of the present study was to adapt the LongSAGE technique for analysis of global gene expression in B. napus and other similarly complex poly- ploid plant genomes where the complete genome sequence and annotation are not yet available. A data processing pipeline was adapted by matching B. napus tags via Brassica ESTs to annotated A. thaliana gene loci, includ- ing detection of tags matching in sense and antisense ori- entation. Results B. napus seed LongSAGE libraries Two LongSAGE libraries were produced from B. napus seeds harvested at 23 and 35 DAP, respectively. A total of 3,136 clones from the 23 DAP library yielded 34,984 dit- ags (22 tags/clone), while 3,168 sequence traces from the 35 DAP library yielded 15,919 ditags (10 tags/clone) (Table 1). The relatively low average number of tags per clone resulted from a high number of cloned empty vector plasmids (28.2% and 60.3% of all high-quality reads for the 23 DAP and 35 DAP libraries, respectively). The vector plasmid pZErO (Invitrogen) was designed to prevent cloning of empty vector by using a LacZa-ccdB fusion cDNA insert that should destroy bacteria containing only the vector. However, as described by other authors (e.g. [12]) we found that pZErO could efficiently re-ligate with- out containing inserts. Thus, PCR screening of plasmid libraries for clones containing an empty vector might con- siderably reduce total Sanger sequencing costs and increase total tag sample sizes in LongSAGE transcription profiling experiments. Table 1 summarises the numbers of accepted and rejected tags in the two libraries. In total, 73,697 tags comprising 37,367 unique tags were extracted from both libraries. Valid tags from 23 DAP and 35 DAP were deposited in the Gene Expression Omnibus (GEO) public domain under accession number GSE14313. The total number of accepted tags from the 23 DAP library was 48,514 com- prising 24,654 unique tags. The total number of accepted tags from the 35 DAP library was 25,183 tags comprising 12,713 unique tags. 67.0% and 64.9% of the accepted total tags were represented by five or less copies in the 23 DAP and 35 DAP libraries, respectively. Matching B. napus tags to the A. thaliana proteome via Brassica ESTs Table 2 summarises the results from the tag-to-gene matching analysis of the two libraries. Initially, all ESTs from a redundant database consisting of 834,732 Brassica EST entries (from the A and C genome brassicas B. napus, B. oleracea, B. rapa) were oriented by blastx alignment against the A. thaliana TAIR7 proteome (cut-off value 1e- 6). Virtual tags were then extracted from all successfully oriented ESTs and assigned via these ESTs to the anno- tated A. thaliana proteins. A total of 3,563,603 virtual 21 bp tags were extracted from all positions of the ESTs. From the 32,395 different observed tags, 15,955 (49.3%) were matched under the applied conditions to Brassica A or C genome ESTs (Table 2). Matching success was reduced about 30% for singleton tags compared to tags with counts ≥ 2. For tags with counts ≥ 2 the matching success Page 2 of 16 (page number not for citation purposes)