Sadd et al. BMC Genomics 2010, 11:110 http://www.biomedcentral.com/1471-2164/11/110 Comparisons to the honeybee and other insects The GC content of the B. terrestris unique sequences was calculated to be 36 percent (omitting ambiguous bases). Gene regions in the A. mellifera genome have a GC content of 29 percent, while in D. melanogaster and A. gambiae it is 44 and 47 percent, respectively [32]. The GC percentage based on these B. terrestris ESTs suggests that lower GC content of gene regions is not restricted to Apis, and may be a wider phenomenon. However, calculations of GC-content in Nasonia vitri- pennis mRNA (version dated 9th July 2007) at 46 per- cent hint that it is not Hymenopteran wide and further species would need to be studied to determine the exact range of this low GC-content. We compared the B. terrestris unique sequences to proteins from genomes of each of four other insects, A. mellifera, N. vitripennis, T. castaneum and D. melanoga- ster (Figure 1, BLASTX, cutoff = 1e-05) (Additional file 5). The hierarchy of “hits” to each of these four insects reflects the evolutionary relationship between them and the bumblebee [32,40,41], with the closest related of the four, A. mellifera, giving 15.9 percent more “hits” than the most distantly realted, D. melanogaster. Despite this, 212 unique sequences from this EST project have signif- icant “hits” to all of these insects except for A. mellifera. Potential explanations for this discrepancy include an incomplete annotation of genes in the A. mellifera gen- ome or loss of genes or their protein coding capacity following the split from Bombus in the lineage leading to Apis (see below). Figure 1 The number of unique Bombus terrestris sequences uncovered in this project with significant “hits” (BLASTX, cutoff = 1e-05) to proteins predicted in each of the Apis mellifera, Nasonia vitripennis, Tribolium castaneum and Drosophila melanogaster genomes (black) and those with no “hits” to the predicted proteins of these organisms (white). Numbers inside bars represent the actual number of unique sequences. Page 4 of 9 The 7,216 “hits” of unique sequences to A. mellifera proteins were to 4,480 unique Apis proteins. This level of redundancy suggests that 62 percent of the assembled sequences are actually derived from unique genes, yield- ing an estimation of 8,278 unique genes represented by this EST library. In addition to a comparison with the predicted proteins in the A. mellifera genome, the unique sequences derived from the B. terrestris EST library were also compared to the entire A. mellifera genome sequence (BLASTN, cutoff = 1e-08) [32]. A total of 10,024 unique sequences had “hits” to the gen- ome, 6,749 of which already had “hits” to predicted A. mellifera proteins, but 3,275 which did not. These unique sequences with “hits” to the genome were dis- tributed on the Apis linkage groups as follows, with the figure in brackets being the percent distribution of actual Apis protein coding sequences: 9.98 (9.57) per- cent on LG1, 5.89 (5.12) on LG2, 4.94 (4.61) on LG3, 4.94 (4.52) on LG4, 6.74 (5.77) on LG5, 3.73 (3.55) on LG6, 3.5 (3.56) on LG7, 5.25 (4.79) on LG8, 3.79 (3.76) on LG9, 5.04 (4.44) on LG10, 6.24 (5.97) on LG11, 3.20 (2.80) on LG12, 3.25 (3.03) on LG13, 4.03 (4.13) on LG14, 4.35 (4.61) on LG 15, 2.77 (2.30) on LG16, 0.02 (0.14) on the Mitochondria and 22.38 (27.34) on unas- sembled regions. Particularly interesting is the apparent discrepancy between the number of B. terrestris unique sequences with matches to A. mellifera proteins and the number with matches to the A. mellifera genome. In fact, of the 212 unique sequences that had “hits” to all the other insect proteins tested but not Apis, 196 have “hits” to the Apis genome. There are a variety of possible reasons for a unique sequence having a “hit” to the Apis genome but not to predicted proteins from the same organism. The most obvious, is that the sequences in question are related to known Apis proteins, but they mainly contain untranslated regions (UTRs) and therefore do not pro- duce significant “hits” in BLASTX alignments. When comparing the locations of the start of “hits” to the gen- ome and the positions of predicted Apis protein coding sequences, 389 are either inside a protein coding sequence or span the boundary of one, suggesting that they comprise mostly of UTRs. Furthermore, 1,064 of the “hits” were within the range of possible Apis UTRs (maximum Apis UTR = 4,375 bases, obtained from the UCSC genome browser [42]). However, this still left 1,253 sequences, including 76 of the unique sequences that had “hits” to proteins from all the other insects tested but not Apis. These 1,253 sequences were further analysed for the presence of ORFs using ORF Finder http://www.ncbi.nlm.nih.gov/gorf. The median ORF size was 225 nucleotides (range 102-1,464) suggesting that many of the unique sequences have the potential to encode proteins. While some of these “hits” to the Apis