the influence of a point was related to its covariance ratio: a covariance ratio larger ( or smaller ) than 1 implied that the point was closer ( or farther ) than was typical to the regression line, so removing it would hurt ( or help ) the accuracy of the line and would increase ( or decrease ) the error term variance. influence was computed as the absolute difference between the covariance ratio and unity. to illustrate further, a point was classified as an “ outlier ” if it had a large studentized residual ( p < 0. 01 ) and low leverage ( boxed point “ a ” ); as a “ high - leverage ” point if it had a low studentized residual and high leverage ( boxed point “ b ” ); and as a “ high - influence ” point if it had a high studentized residual, high leverage, and high influence ( boxed point “ c ”; figure 4b ). points that resembled boxed point “ a ” were designated as potential as events. for example, four of the five boxed points in figure 3c were “ outliers, ” and rt - pcr validation indicated that the exon represented by the probeset was indeed skipped in hcns - scns ( ehbp1, figure 7b ). points that were “ high - leverage, ” such as the five points in the clcn2 gene, were experimentally verified to be a fp ( figure 4c; unpublished data ). points that were “ high - influence, ” such as the four of five boxed points in the abca3 gene were also experimentally verified to be a fp ( figure 4d; unpublished data ). in conclusion, in order to reduce the fp rate, all points were evaluated according to the metrics described, and points that were significant “ outliers ” were considered putative as events. figure 4sources of false positives ( a ) scatter plot of points for the fip1l1 gene and the line representing the robust regression estimate. ( b ) boxed point “ a ” represents a significant “ outlier ” ( with a significantly different studentized residual and low leverage ). boxed point “ b ” represents a “ high leverage ” point ( low studentized residual and a high leverage ). boxed point “ c ” represents a “ high influence ” point ( high studentized residual, high leverage, and high influence ). ( c ) scatter plot of points for the clcn2 gene. boxed points represent “ high leverage ” points. ( d ) scatter plot of points for the abca3 gene. boxed points represent “ high influence ” points. global identification and characterization of reap [ + ] exonsreap was applied to identify as events in nps compared to hescs: cyt - np versus cyt - es; hues6 - np versus hues6 - es; hcns - scns versus cyt - es, and hcns - scns versus hues6 - es. after removing potential fps, 11, 348 genes containing 158, 657 probesets were scored by reap. as described above, for each pair of cell lines compared, each probset was represented by five points, where each point was defined a significant outlier if it had a high residual ( p < 0. 01 ), low influence, and high leverage. points per probeset should be correlated; in other words, if one point was a significant outlier, the other points were expected to be outliers as well. to ensure that this was the case, we counted the number of probesets with n significant outliers, where n was varied from 0 to 5. next, the identity of the probesets and points derived from them were exchanged with other probesets, keeping constant the total number of points that were considered significant outliers. at n = 0, we observed approximately equal numbers of probesets in the actual versus shuffled controls. in contrast, we observed that there were 1. 5 times more probesets with n = 2 significant outliers relative to shuffled controls; 12 – 31 times more probesets with n = 3; and 17 – 612 times more probesets that had n = 4 significant outliers ( figure 5a; see table s1 ). for example, in hcns - scns compared to cyt - es, approximately 0. 39 % ( 490 of 124, 604 ) of probesets had three significant outliers and 0. 25 % ( 308 probesets ) had four significant outliers, relative to 0. 02 % and 0 % of shuffled controls, respectively. figure 5correlation between “ outliers ” ( a ) the number of probesets with n significant “ outliers ” was determined for hcns - scns versus cyt - es, hcns - scns versus hues6 - es, cyt - nps versus cyt - es, and hues6 - nps versus hues6 - es ( n = 0, 1, 2, 3, 4, 5 ). for comparison, points to probeset relationships were randomly permuted, retaining the same number of “ outliers. ” vertical bars represent the ratio between the number of actual points and the randomly per mutate d sets. ( b ) similar to ( a ), except points were counted as “ outliers ” only if they were “ outliers ” in both hcns - scns versus cyt - es and hcns - scns versus hues6 - es ( combined hcns - scns versus hesc; blue bars ); in both hues6 - np versus hues6 - es and cyt - np versus cyt - es ( combined derived np versus hesc; red bars ); and in all four comparisons ( combined np versus hesc; yellow bar ). next we asked whether the overlap between related comparisons was higher than expected. comparing the significant probesets between hcns - scns versus cyt - es and hcns - scns versus hues6 - es revealed 672 significant probesets ( n ≥ 2 ), whereas if we shuffled the associations between probeset identity and significant outliers, only four significant probesets ( n ≥ 2 ) were identified — a 168 - fold enrichment ( figure 5b, table s1 ). a total of 236 significant probesets overlapped when we compared the derived nps to hescs ( cyt - np versus cyt - es and hues6 - np versus hues6 - es ), relative to seven significant probesets ( 34 - fold enrichment ). at a cutoff of two significant outliers, 1, 737 probesets contained in internal exons were defined as positive reap predictions ( hereafter called reap [ + ] ) exons — candidate as events that disting uished np from hesc. surprisingly, we observed that the majority of reap [ + ] exons were specific to the pair of hesc and np that was compared, likely reflecting differences in genetic origins and / or culturing and differentiation conditions of the cell lines: 614 reap [ + ] events were unique to hcns - scns versus hue6 - es; 220 were unique to hcns - scns versus cyt - es; 439 were unique to hues6 - np versus hues6 - es; and 250 were unique to cyt - np versus cyt - es. the shared events between pairs of comparisons made up a minority of the total number identified: 102 reap [ + ] events were found to be in common between hcns - scns versus cyt - es and hcns - scns versus hues6 - es; 48 between hcns - scns versus hues6 - es and hues6 - np versus hues6 - es; and only 17 between hcns - scns versus cyt - es and cyt - np versus cyt - es ( table s2 ). comparison of reap to est - based method and acescantraditionally, as exons were discovered by using est alignments to genomic loci, and also more recently by computational algorithms that used sequence information extracted from multiple genomes. here, we compared reap predictions to both approaches. in the first comparison, publicly available ests and mrna transcripts were aligned to the human genome sequence. 13, 934 exons with evidence for exon - skipping and / or inclusion ( est - se for est - verified skipped exons ) were generated, comprising ∼7 % of all internal exons. first we analyzed cyt - es versus hcns - scns. if we required that none of the points per probeset ( exon ) was significant, 6 % ( 4, 402 of 71, 731 ) of exons ( after probeset mapping ) had evidence for est - se ( figure 6a ). shuffling the mapping between these probesets and exons resulted in 8 % ( 5, 777 of 71, 731 ) of exons with evidence for est - se ( figure 6a ). these percentages were not significantly different from the 7 % of exons with est evidence for as observed from using all exons. by raising the requirement that probesets had to contain at least one significant point to five significant points, the percentage of est - se increased dramatically from 11 % ( 531 of 4, 898 exons ) to 26 % ( 33 of 126 ).