the number of true negatives ( tn; or fps ) was computed as the number of reap [ − ] ( reap [ + ] ) exons that were validated by rt - pcr as constitutively spliced. the true ( false ) positive rate was computed as tp ( fp ) divided by the total number of reap [ + ] exons in the experimentally validated set. the true ( false ) negative rate was computed as the tn ( fn ) divided by the total number of reap [ − ] exons in the experimentally validated set. sensitivity was computed as tp / ( tp + fn ) and specificity was computed as tn / ( fp + tn ). genome sequences of human ( hg17 ), dog ( canfam1 ), rat ( rn3 ), and mouse ( mm5 ) were obtained from ucsc, as were the whole - genome multiz alignments [ 80 ]. the lists of known human genes ( knowngene containing 43, 401 entries ) and known isoforms ( knownisoforms containing 43, 286 entries in 21, 397 unique isoform clusters ) with annotate d exon alignments to human hg17 genomic sequence were processed as follows. known genes that were mapped to different isoform clusters were discarded. all mrnas aligned to hg17 that were greater than 300 bases long were clustered together with the known isoforms. genes containing less than three exons were removed from further consideration. a total of 2. 7 million spliced ests were mapped onto the 17, 478 high - quality genes to infer as. exons with canonical splice signals ( gt - ag, at - ac, gc - ag ) were retained, resulting in a total of 213, 736 exons. of these, 197, 262 ( 92 % of all exons ) were constitutive exons, 13, 934 exons ( 7 % ) had evidence of exon - skipping, 1, 615 ( 1 % ) exons were mutually exclusive alternative events, 5, 930 ( 3 % ) exons had alternative 3 ′ splice sites, 5, 181 ( 2 % ) exons had alternative 5 ′ splice sites, and 175 ( < 1 % ) exons overlapped another exon, but did not fall into the above classifications. a total of 324, 139 probesets from the affymetrix human exon 1. 0 st array were mapped to 208, 422 human exons, representing 17, 431 genes. these probesets were used to derive gene and exon - level signal estimates from the cel files. the four - way mammalian ( four - mammal ) whole - genome alignment ( hg17, canfam1, mm5, rn3 ) was extracted from the eight - way vertebrate multiz alignments ( hg17, pantrol1, mm5, rn3, canfam1, galgal2, fr1, danrer1 ) obtained from the ucsc genome browser. four - way mammal alignments were extracted for all internal exons, and 400 bases of flanking intronic sequence, resulting in a total of 161, 731 conserved internal exons. a total of 145, 613 ( 90 % of total ) conserved internal exons were constitutive exons, 13, 653 exons ( 8 % ) had evidence of exon - skipping, 1, 576 exons were mutually exclusive alternative events, 5, 818 exons had alternative 3 ′ splice sites, 5, 046 exons had alternative 5 ′ splice sites, and 168 exons overlapped another exon. the affymetrix power tools ( apt ) suite of programs was obtained from http: / / www. affymetrix. com / support / developer / powertools / index. affx. exon ( probeset ) and gene - level signal estimates were derived from the cel files by rma – sketch normalization as a method in the apt - probeset - summarize program. to determine if the signal intensity for a given probeset is above the expected level of background noise, we utilized the dabg ( detection above background ) quantification method available in the apt - probeset - summarize program as part of affymetrix power tools ( apt ). briefly, dabg compared the signal for each probe to a background distribution of signals from anti - genomic probes with the same gc content. the dabg algorithm generated a p - value representing the probability that the signal intensity of a given probe was part of the background distribution. we considered a probeset with a dabg p - value lower than 0. 05 as detected above background. the statistic thcns - scns, esc = ( μhcns - scns − μesc ) / sqrt ( ( ( nhcns - scns − 1 ) σ2hcns - scns + ( nesc − 1 ) σ2esc ) ( nhcns - scns + nesc ) ) / ( ( nhcns - scnsnesc ) ( nhcns - scns + nesc − 2 ) ) ), where nhcns - scns and nesc were the number of replicates, μhcns - scns and μesc were the mean, and σ2hcns - scns and σ2esc were the variances of the expression values for the two datasets used to represent the differential enrichment of a gene using gene - level estimates in hcns - scns relative to hescs. multiple hypothesis testing was corrected by controlling for the false discovery rate ( benjamini - hochberg ). as detection by reap. the log2 signal estimate xij for probeset i in cell - type j had to satisfy two conditions, otherwise the probeset was discarded: ( i ) 2 < xij < 10, 000 for all conditions / cell - types j; and ( ii ) dabg p - value < 0. 05 for all replicates in at least one condition / cell - type j. a gene had to have five probesets that satisfied the two conditions above in order to be considered for robust regression analysis. after generating the points ( as described in the results section ), we utilized the robust regression method rlm in r - package “ mass ” ( version 6. 1 – 2 ) with m - estimation and a maximum iteration setting of 30 to estimate the linear function yi = αxi + β. for each probeset, we computed the error term ei,, which was the difference between the actual value yi and the estimated value ξi, from the estimated function ξi = axi + b, where a and b were estimates of α and β. the error term variance was estimated by se2 = σei2 / ( n − p ), which was used to estimate the variance of the predicted value, sξi2 = se2 ( n−1 + ( xi − μx ) 2 / sx2 ( n − 1 ) ). here, n referred to the number of points ( generated for each gene ), and p referred to the number of independent variables ( p = 2 in our method ); and μx = σxi2 / n; sx2 = n−1 σ ( xi − μx ) 2. following belsley et al. [ 81 ], we defined the leverage hi of the ith point as hi = n−1 + ( xi − μx ) 2 / sx2 ( n − 1 ). here we considered a point to have high leverage if hi > 3p / n. next, we calculated the covariance ratio, covi = ( si2 / sr2 ) p / ( 1 − hi ), which is the ratio of the determinant of the covariance matrix after deleting the ith observation to the determinant of the covariance matrix with the entire sample. we considered a point to have high influence if | covi − 1 | > 3p / n. lastly, we computed the studentized residuals, rstudenti = ei / ( s ( i ) 2 ( 1 − hi ) 0. 5 ), where s ( i ) 2 = ( n - p ) se2 / ( n - p - 1 ) – ei2 / ( n - p - 1 ) ( 1 − hi ), the error term variance after deleting the ith point. as rstudenti was distributed as student ' s t - distribution with n - p - 1 degrees of freedom, each rstudenti value was associated with a p - value. we considered a point to be an “ outlier ” if p < 0. 01. the enrichment score of a sequence element of length k ( k - mer ) in one set of sequences ( set 1 ) versus another set of sequences ( set 2 ) was represented by the nonparametric χ2 statistic with yates correction, computed from the two by two contingency table, t ( t11: number of occurrences of the element in set 1; t12: number of occurrences of all other elements of similar length in set 1; t21: number of occurrences of element in set 2; t22: number of occurrences of all other elements of similar length in set 2. all elements had to be greater than 5. to correct for multiple hypothesis testing, p - values were multiplied by the total number of comparisons. this work uncovers novel mechanisms of aging within stem cell niches that are evolutionarily conserved between mice and humans and affect both embryonic and adult stem cells. specifically, we have examined the effects of aged muscle and systemic niches on key molecular identifiers of regenerative potential of human embryonic stem cells ( hescs ) and post - natal muscle stem cells ( satellite cells ).