BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 FAigscuartete3r plot showing the number of conserved TPAs on each human chromosome versus the chromosome size (in Mb) A scatter plot showing the number of conserved TPAs on each human chromosome versus the chromosome size (in Mb). The chromosome X is circled. The collective size of the human genome, excluding chromosome X, is 3,098,124,053; and the size of the chromosome X after normalizing for chromosome dosage in gametes, as done by Emerson et al. [30], is 116,185,316 (i.e., 0.75 times the original size of 154,913,754), which harbors 13 transcribed preserved pseudo- genes. Since the collective genome size is 27 times bigger than that of the human chromosome X, we expect 2 or 3 preserved pseudogenes on the chromosome X. Presence of 13 cases, an increase of more than 4.5 times than expected, suggests a statis- tically significant overrepresentation (exact binomial test of goodness-of-fit, P-value < 10-5, for the hypothesis (H0) that there is no difference between the observed and expected frequencies of transcribed preserved pseudogenes on the chromo- some X). does not have any influence on the observed GC content differences. Ka/Ks trends for TPAs that are conserved in rhesus monkey We decided to assess the genomic evidence for a lack of protein-coding ability in the human TPAs that are synten- ically conserved in rhesus monkey. We compared TPA characteristics to the characteristics of two other group- ings: (i) known human protein-coding genes with orthologs in rhesus monkey; (ii) populations of simu- lated sequences that are randomly mutating without cod- ing-sequence selection pressures. The human TPA sequences are used as starting sequences for these simula- tions. The protocol for these simulations is described in 'Methods: Ka/Ks ratio calculations'. The ratio of non-synonymous to synonymous substitu- tion rates (Ka/Ks) provides a measure of selection pressure for protein-coding ability on nucleotide sequences. Clas- sically, values significantly <<1.0 indicate purifying selec- tion, whereas sequences without coding ability theoretically yield values near 1.0. We examined the trends for Ka/Ks in the population of human TPAs that are conserved syntenically in the rhesus monkey. Ka/Ks was calculated using the PAML package (as described in Meth- ods). The distribution of Ka/Ks is shown for TPAs, split into two groups, those that have a disrupted protein domain of known three-dimensional structure (TPADD, blue bar, Figure 5), and those that do not (TPANDD, red bar, Figure 5). In addition, we calculated the distribution of Ka/Ks values for sequences that are randomly mutating without cod- ing-sequence selection pressures. These sequences were generated using the simulation protocol described in 'Methods: Ka/Ks ratio calculations', using the human TPAs as starting sequences (green curve, Figure 5). The Ka/Ks dis- tribution for these simulated sequences does not peak at ~1.0, as would be classically expected. This is likely due to some inaccuracy in modeling the expected frequency for the different possible nucleotide substitutions, which var- ies for different genomic areas [3]. The distribution for Page 6 of 14 (page number not for citation purposes)