BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 FAigscuhreem4atic representation of the procedure for identifying syntenic noncoding (flanking) regions A schematic representation of the procedure for identifying syntenic noncoding (flanking) regions. Flanking regions (10000 nts) are scanned in a sliding window, equal to the length of conceptual human pseudogene transcript), by glo- bally aligning it to the upstream/downstream regions of the human pseudogene. The best scoring window is identified, which corresponds to the syntenic (flanking) region in the monkey genome. TPADDs peaks in the range 0.6 to 1.0. This peak is similar for the randomly-mutating sequences (Figure 5). For the TPANDDs, the peak is at lower Ka/Ks values (0.4-0.6). As a further comparison, we have calculated the Ka/Ks curve for orthologous pairs of protein-coding genes from the rhesus monkey and the human (blue curve, Figure 5). Clearly, these protein-coding sequences behave very dif- ferently from the TPAs, with a substantial mode in the range 0.0 to 0.2. In summary, these Ka/Ks trends indicate that the substitution patterns in the TPAs generally behave like non-protein-coding sequences, and not like protein- coding ones. This is despite the overall significant conser- vation relative to surrounding intergenic genomic DNA that was discussed in the previous section. Analysis of the ratio of non-synonymous to synonymous substitution rates (Ka/Ks) relative to orthologous TPAs in dog and in mouse To gain a more complete picture, we also examined Ka/Ks values for TPAs that are conserved in two more divergent species, the dog and the mouse. We compared Ka/Ks val- ues for orthologous TPA pairs (termed Ka/KsΨ-ortho), with the corresponding Ka/Ks values for their parent genes (Ka/ Ksparent-ortho) (Figure 6). These were calculated for human/ dog (Figure 6(a)), and human/mouse comparisons (Fig- ure 6(b)). For human/dog comparisons, the substantial majority (83%) have Ka/KsΨ-ortho > Ka/Ksparent-ortho, whereas for human/mouse all of the pseudogene pairs have larger Ka/Ks values than their corresponding parent pairs. Table 2: Proportions of pseudogenes that have GC contents higher relative to their flanking regions in different categories of VEGA annotated human pseudogenes. Pseudogene category Transcribed and conserved (monkey) pseudogenes Non-transcribed pseudogenes P-value for statistical difference* Processed Nonprocessed Unclassified Total * χ2 test with d.f. = 1. 165/188 (87.77%) 9/14 (64.29%) 153/189 (80.95%) 327/391 (83.63%) 2473/3274 (75.53%) 577/910 (63.40%) 2239/2970 (75.39%) 5289/7154 (73.93%) < 10-04 0.7807 0.0961 < 0.0001 Page 7 of 14 (page number not for citation purposes)