Figure 3

Hybridization to probe sets on the MuscleChip provides verification of singleton, and other low member EST clusters. Panel A. Graph output from "the Vertical Line" program showing unique EST singletons or clusters (duplex, or triplex) as individual lines emanating from the left axis (777 clusters total). Probe sets were designed for each cluster on the MuscleChip, and normal muscle cRNA hybridized to the MuscleChip. Using default Affymetrix algorithms, hybridization pattern to each probe set were assigned as "present" (red lines), "marginal" (yellow lines), or "absent" (black lines) (See Methods). There is a clear correlation with definition of "present" calls and EST cluster member number (see also Table 3). This result shows that 30% of EST singletons were verified as "present" in normal muscle RNA by this method. Panel B. Shown is a subset of EST that showed no match to a known gene, when analyzed by BLAST (Table 4). As in Panel A, "the Vertical line Program" is used to map the absolute intensity to clone number in EST cluster, using the default Affymetrix algorithms and hybridization pattern to assign each probe set a call as "present" (red lines), "marginal" (yellow lines), or "absent" (black lines). Out of 250 ESTs, 195 EST clusters had 2, 3 or 4 EST clone members. We found 81% of quadruple clusters to be "present" or "marginal", 72% of triplex clusters, and 45% of duplex showing increase in "present " calls with clone number.