Measuring Selective Pressure on Each RESCUE-ESE Hexamer

<div><p>Any point mutation alters six overlapping hexamers, and so a database of 8,408 SNP mutations alters a total of approximately 50,000 hexamers in the wild-type (ancestral) allele. In considering all 238 RESCUE-ESE hexamers, the frequency of each ESE hexamer in the total set of ancestral alleles was recorded for the database of SNPs and simulated mutations (8,408 SNP mutations and 100,000 simulated mutations). The ESE frequency in the SNP set was divided by the ESE frequency in the simulated set to calculate the RR for each of the 238 hexamers.</p> <p>(A) The distribution of RR for all 238 ESE hexamers is plotted on a logarithmic scale. A resampling strategy was used to identify 57 ESE hexamers that were significantly conserved (pink bars have an RR less than 1; <i>p</i> < 0.05) and also six ESE hexamers that were not conserved (blue bars have an RR greater than 1; <i>p</i> < 0.05).</p> <p>(B) The output of RESCUE-ESE was compared for several vertebrate genomes (human, mouse, pufferfish, and zebrafish). The set of 238 human RESCUE-ESE hexamers was divided into nonoverlapping subsets based on their conservation in the RESCUE-ESE output generated from other vertebrates. The proportion of ESEs that were significantly conserved in the SNP analysis (as described above in [A]) were recorded for each subset of RESCUE-ESE hexamers and are represented as pink sectors in the pie chart.</p></div

Density of Predicted ESEs and SNPs along Human Exons

<p>RESCUE-ESE hexamers were searched against a database of 121,000 internal human exons. ESE density (blue curve) was determined as the fraction of hexamers beginning at the given exon position in this dataset that were contained in the RESCUE-ESE set. SNP density (red curve) was determined analogously using SNPs from dbSNP mapped to the exon database. Both curves were smoothed by averaging the densities over a leading (3′ss) or lagging (5′ss) window of ten nucleotides.</p

Selection against Disruption of Predicted ESEs in Different Exon Regions

<p>Summary ORs were calculated for mutations that disrupt RESCUE-ESEs as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020268#pbio-0020268-g002" target="_blank">Figure 2</a>, for each of four regions spanning the length of a typical human internal exon. The heights of the blue bars represent the odds that an ESE will be disrupted by a mutation in the set of 2,561 validated SNPs (selected mutations) relative to the odds of disruption in the set of 100,000 simulated (unselected) mutations. Error bars extend one standard deviation on either side of the calculated value.</p