Page 35 - Biosystems Engineering

16 Cha pte r O n e

FIGURE 1.8 A scanned Affymetrix GeneChip.

Figure 1.7 illustrates Affymetrix GeneChip design. Figure 1.8 shows
the appearance of a scanned Affymetrix GeneChip.

1.4 Low-Level Analysis
cDNA microarray data preprocessing involves image quantification,
quality control, background correction, and normalization. Image
quantification involves several steps: (1) finding where the printed
spots are, (2) identifying the extent of each probe and separating fore-
ground from background, (3) summarizing the varying brightness of
the pixels in the foreground of each spot, (4) dealing with scanner
saturation, and (5) dealing with variable backgrounds. Background
correction is done by either subtracting the estimated local background
or by subtracting values of negative controls nearby. Negative con-
trols are probes designed for DNA sequences that should never occur
in the sample. These controls should read low signals and should be
fairly uniform. These signals give an estimate of the background due
to nonspecific hybridization. Positive controls are replicate probes for
sequences that should be abundant and are used to estimate the spa-
tial variation in hybridization. Spike-in controls are probes for tran-
scripts not expected in the sample but added to the sample with
known concentration. These controls give an estimate of the accuracy