a) Number of genes investigated with different sets of reference genes. Bar heights indicate the number of genes which were surveyed with tag profiling (TP, light shading) and a previous microarray study (MI, dark shading). With the small reference sets, increasing the number of mismatches in mapping Pachycladon tags to Arabidopsis TAIR10 protein coding sequences increased the number of investigated genes, but did not reach the number of genes investigated when mapping Pachycladon tags to P. fastigiatum full-length ESTs. Clearly, more genes were investigated when tags were mapped against all contigs of the P. fastigiatum EST library or the entire TAIR10 collection. For abbreviations of different sets of reference genes see Additional file 3. b) Number of differentially expressed genes (DEGs) in P. enysii and P. fastigiatum with different sets of reference genes. The number of DEGs identified in each species is correlated with the total number of genes amenable to study for differential gene expression (compare Figure 2a and Figure 2b). The percentages of up-regulated genes in relation to the investigated gene sets ranged from 6-18% in P. fastigiatum and from 9-22% in P. enysii. Except for reference sets PL0 and AL2, more DEGs were identified in P. enysii than in P. fastigiatum.