Wang et al. Genetics Selection Evolution 2010, 42:24 http://www.gsejournal.org/content/42/1/24 duplicates have been created via small-scale events in mammalian genomes [8]. In addition, we made the assumption that the duplication rate for each type is con- stant per year rather than per generation in mammalian genomes. This seems a reasonable assumption because the duplication rate has often been presented with respect to absolute time scale in previous studies [9,10]. Methods Collection of duplicates All paralogs (protein-coding genes with pseudogenes excluded) and relevant annotations (identity scores, loca- tions and exons) were retrieved from Ensembl database (release 50) via BioMart [11], which amounted to 80,683 and 159,047 pairs of duplicates in human and mouse genomes, respectively. The original dataset had a lot of redundant pairs in multi-member gene families. For example, in a n-member family, there would be n(n-1)/2 paralogous pairs listed, although at most only n-1 dupli- cation events were needed to create the family. In this case, we only chose the n-1 pairs that contained all the members and had the highest total identity score. Alto- gether, 9,425 and 11,224 non-redundant pairs were pre- served for the two genomes. Next, we applied CHSMiner [12] to detect and remove paralogous segments arising from large-scale duplications. The segments should con- tain at least two pairs of duplicates, and the gap size between two neighbouring duplicates in either segment should be less than 30 genes [13]. The duplicated pairs located in those segments with FDR < 0.05 were filtered. After this step, we obtained 6,552 and 8,308 pairs for fur- ther screening in human and mouse genomes, respec- tively. Screening tandem duplicates and retrogenes Although tandem duplicates should be adjacent to each other on one chromosome, the extensive gene inversions may insert irrelevant genes into the tandem arrays. We followed the stringent definition adopted by previous studies [14,15] to screen the tandem duplicates, which restricted the inserted spacers to no more than one gene. This resulted in 1,210 and 1,802 paralogous pairs in human and mouse genomes, respectively [see additional file 1 and 2]. We implemented a method similar to those of Emerson [16] and Pan [15] to screen retrogenes. First, the pairs with a multi-exon member and an intronless member were considered as putative parental-retrogene pairs, but the pairs with both members intronless were ignored as they were not clearly created via retroposi- tions. Next, for the putative pairs with both members located on the same chromosome, we discarded those with the intervening spacers containing less than 10 genes, since they were confused with tandem duplicates. Finally, we preserved 410 and 680 pairs resulting from Page 2 of 6 retropositions in the two genomes, respectively [see addi- tional file 3 and 4]. Sequence similarity analysis The similarity of protein sequences between two dupli- cates, as measured by their average amino acid identity, can be retrieved directly from BioMart. The dN and dS of their coding sequences were downloaded from the EPGD database http://epgd.biosino.org/EPGD/[17] [see addi- tional file 1, 2, 3, and 4]. To avoid the influence of satura- tion effect [18], only the pairs with dS < 1 were considered in the dN/dS analysis. Expression similarity analysis The tissue-specific expression profiles and the annotation of the probesets were downloaded from the GNF gene expression database http://wombat.gnf.org[19]. We chose the datasets HUMAN U133A/GNF1H and MOUSE GNF1M for the corresponding species. The Present/ Absent calls in the profiles were used to indicate whether a probeset was expressed or not, and the Marginal calls were also treated as Present calls. When a gene had many probesets, it was considered to be expressed if any one of the probeset was present. We ignored the probesets such as '_f_at', '_s_at' and '_x_at' because they could not be mapped to unique genes in a gene family. For a duplicated pair, common probesets shared by the two members were also excluded. Finally, if s was the number of tissues where both members were expressed, and d was the number of tissues where one member was expressed while the other was not, then their expression similarity was calculated as s/(s+d) [see additional file 1, 2, 3, and 4]. Results Gene duplicability We identified 1,210 tandem duplicates and 410 retro- genes in the human genome, and 1,802 tandem duplicates and 680 retrogenes in the mouse genome. The higher number of tandem duplicates than retrogenes in both genomes implies a higher gene duplicability for tandem duplicates. Previous studies have found that gene dupli- cability is positively correlated with gene dosage [20] and gene complexity [21], although the correlation with func- tional essentiality is not always the same in yeasts and mammals [22-25]. To investigate the difference in gene duplicability between tandem duplicates and retrogenes in more detail, we counted the number of each type of duplicates in gene families with various sizes (Figure 1). The result shows that their distributions among gene families are quite different (p < 0.01 for both genomes, chi-square test). Specifically, tandem duplicates are more likely to be enriched in larger families, whereas retro- genes do not display a preference.