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Fine Tuning Your Data Analysis:,,, Tunable Parameters of the Affymetrix Expression Analysis Statistical Algorithms

enerated based on limits of confidence set for detection. The cutoffs alpha1 and alpha2 determine the boundaries between the three possible calls. As alpha1 and alpha2 are increased or decreased, the range of p-values that is interpreted as a P, M or A call will change. Present calls are output for probe sets when the Detection p-value is below the parameter alpha1. An Absent call is output if the p-value is greater than alpha2. The call is Marginal if the p-value lies between the two thresholds. Optimal default values were established (for 16-20 probe pair designs) at alpha1 = 0.04 and alpha2 = 0.06.

Increasing alpha1 to levels above the default relaxes the stringency by allowing genes with higher Detection p-values to be called Present. This effect is illustrated in Figure 2, which shows how the percentage of probe sets scoring Present calls changes across the entire array as alpha1 is increased. A high alpha setting permits more transcripts to score Present; such a setting provides a higher level of sensitivity, but comes at the cost of a greater level of false positives. Conversely, decreasing alpha1 below the default causes a decline in the percentage of Present calls made, leading to fewer false positives at the cost of losing some truly expressed genes.

The tradeoff between detection sensitivity and specificity is shown in more detail in Figure 3. The scatter plot shows the relationship of the alpha cutoffs to probe set data displayed by Signal (y-axis) and Detection p-value (x-axis). Optimal placement of the alpha cutoffs provides a balance between true detection and false positives. Setting alpha cutoffs to higher or lower p-values alters the call stringency. An example is shown with the dashed red line, representing alpha1 set to 0.01. This highly stringent setting decreases false detection to near zero levels, at the cost of losing true positives (35% Present at default alpha1 declines to 26% at the altered se
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