in comparison, the shuffled probesets at the same requirements remained at ∼8 %, rising slightly to 11 % at five points, due to small sample sizes. similar trends were observed with hcns - scns versus hues6 - es and the derived nps versus hescs ( figure 6a ). therefore, we concluded that reap [ + ] exons were enriched for as events independently identified by a transcript - based approach. figure 6comparison of reap predictions for hcns - scns versus cyt - hes, hcns - scns versus hues6 - es, cyt - np versus cyt - es, and hues6 - nps versus hues6 - es with alternative exons identified by an est - based method and acescan ( a ) black - filled squares represented the fraction of exons containing probesets with n significant points that had est evidence for exon inclusion or exclusion ( n = 0, 1, 2, 3, 4 and 5 ). white - filled triangles represented similarly computed fractions with permuted probeset to exon mappings. ( b ) black - filled squares represented the fraction of exons containing probesets with n significant points that had acescan positive scores, indicative of evolutionarily conserved alternative exons. white - filled triangles represented similarly computed fractions with permuted probeset to exon mappings. next, we compared reap predictions to a computational approach of identifying exons with as conserved in human and mouse, acescan [ 55 ]. acescan receives as input orthologous human – mouse exon pairs and flanking intronic regions and computes sequence features and integrates the features into a machine - learning algorithm to assign a real - valued score to the exon. a positive score indicated a higher likelihood of being as in both human and mouse. acescan was updated in the following ways. firstly, instead of relying on orthology information by ensembl, and then aligning flanking introns in “ orthologous ” exons, conserved exonic and intronic regions in human and mouse from genome-wide multiple alignments were extracted. secondly, whereas in our previous analysis exons from the longest transcript in ensembl were utilized, now we collapsed all the transcripts available at the ucsc genome browser and analyzed all exons in the entire gene loci. acescan was utilized to assign acescan scores to all ∼162, 000 internal exons in our genes. exons annotate d as first or last exons in refseq mrnas were excluded from our analysis, resulting in 4, 487 positive - scoring exons, 2 - fold more exons than originally published. here we repeated our analysis with exons with positive acescan scores ( ace [ + ] ) instead of est - ses. if we required that none of the points per probeset ( exon ) was significant, 2 % ( 1, 645 of 71, 731 ) of exons ( after probeset mapping ) were ace [ + ] ( figure 6b ). shuffling the mapping between these probesets and exons resulted in 3 % ( 2, 044 of 71, 731 ) of exons being ace [ + ] ( figure 6b ). these percentages were not significantly different from the 2. 7 % observed from all exons ( 4, 487 of the 162, 000 exons that were scored by acescan ). by raising the requirement that probesets had to contain five significant points, the percentage of ace [ + ] exons increased from 4 % to 11 %. however, the sample sizes were small. in comparison, the shuffled probesets at the same requirements remained at ∼4 %. similar overall trends were observed with hcns - scns versus hues6 - es and the derived nps versus hescs ( figure 6b ). in total, 7. 5 % ( 131 of 1, 737 ) of reap [ + ] exons were designated as acescan [ + ] compared to 2. 4 % ( 2, 328 of 97, 437 ) of reap [ − ] exons. this result suggested that a small but significantly enriched fraction of as events in hescs versus nps was likely to be evolutionarily conserved in human and mouse. in conclusion, our results suggested that reap predictions were congruent with predictions from two independent, orthogonal methods. experimental validation of alternative exonsthe sensitivity and specificity of reap in the identification of reap [ + ] exons was tested by rt - pcr. to validate reap [ + ] alternative exons, rt - pcr primers were designed in the flanking exons to amplify both isoforms. to be a positively validated candidate, the pcr products on a gel had to satisfy all of the following criteria: ( i ) at least one isoform with the expected size must be visible in each cell type; ( ii ) the relative abundance of the two isoforms must be altered between two cell types and the direction of change have to be consistent with the reap studentized residuals: in our study positive residuals implied inclusion in hescs and skipping in nps, and negative residuals implied inclusion in np and skipping in hescs; and ( iii ) the results were replicable in at least two experiments. for simplicity of design, we tested candidates predicted from cyt - es versus hcns - scns. fifteen reap [ + ] exons with at least two significant outliers ( out of five ) were randomly chosen as predicted alternative events and thirty - five exons with less than two significant outliers were randomly chosen as constitutive events ( table s3 ). nine of the fifteen exons ( 60 % ) were validated as as events by our criteria. the sensitivity and specificity of the algorithm at the cutoff of two is 69 % and 77 %. increasing the cutoff to three increased the specificity to 85 %, with a slight decrease in sensitivity to 67 % ( figure 7a ). the patterns of as in hescs were similar in both cyt - es and hues6 - es for all as events validated, but the nps ( cyt - np, hues6 - np, and hcns - scns ) had more varied as. the pattern of as in the reap [ + ] exons in the slk ( serine / threonine kinase 2 ) and pot1 ( protection of telomeres 1 ) genes showed remarkable agreement within derived nps and hcns - scns ( figure 7b ). the as exon in slk was observed to be included in hescs and completely excluded in nps; the as exon in the pot1 gene was included more in hescs and a smaller isoform persisted in nps. the as patterns of the other verified reap [ + ] exons were consistently similar in hescs but were more varied in the np. interestingly, the patterns of as in the derived nps ( cyt - np and hues6 - np ) were not always identical to those of hcns - scns. for example, the as exon in the ehbp1 ( eh domain binding protein 1 ) gene was included in hescs but skipped in hcns - scns, and both isoforms were present in the derived nps ( figure 7b ). as another example, the as exon in the sorbs1 ( sorbin and sh3 domain containing 1 ) gene was skipped in hescs and included in hcns - scns, but exhibited an intermediate pattern in the derived nps. however, in some cases, the as patterns in the derived nps were different from both hescs and hcns - scns ( such as in the as exon in unc84a, sirt1, and mllt10 ). figure 7rt - pcr validation of reap - predicted alternative exons ( a ) probesets ( exons ) were considered reap [ + ] candidates if they contained at least n = 2 ( white bars ), 3 ( gray bars ), or 4 ( black bars ) significant outliers. true positive ( tp ), true negative ( tn ), false positive ( fp ), and false negative ( fn ) rates were calculated from rt - pcr - validated reap [ + ] exons at the different cutoffs ( n = 2, 3, 4 ). ( b ) nine rt - pcr validated reap [ + ] as events in hescs ( cyt - es and hues6 - es ), derived nps ( cyt - np and hues6 - np ), and hcns - scns. arrows indicate the larger ( exon - included ) isoforms and smaller ( exon - skipped ) isoforms. ( c ) rt - pcr of reap [ + ] alternative exons from ehbp1, slk, and rai14 across a panel of human tissues. arrows indicate the larger ( exon - included ) isoforms and smaller ( exon - skipped ) isoforms. first, given three independent samples each from two conditions, we concluded that reap was able to identify as events with high specificity but with moderate sensitivity.