Calibration and assessment of channel-specific biases in microarray data with extended dynamical range

Henrik Bengtsson, Göran Jönsson and Johan Vallon-Christersson

Centre for Mathematical Sciences
Mathematical Statistics
Lund Institute of Technology,
Lund University,

ISSN 1403-9338
By scanning the same spotted oligonucleotide microarray at different photomultiplier tube (PMT) gains we have identified a channel-specific bias present in two-channel microarray data. The observed bias was very stable between subsequent scans of the same array although the PMT gain was greatly adjusted. This indicates that the bias does not originate from a step preceding the scanner detector parts. Between arrays the estimated bias varies slightly. When comparing bias estimates based on data from the same array, but from different scanners, we have found that different scanner models introduce different amounts of bias. So do various image analysis methods. We propose a scanning protocol and a constrained affine model that allows us to identify and estimate the bias in each channel. Backward transformation removes the bias and brings the signals to the same scale. The result is that systematic effects such as intensity dependent log-ratios are removed, but also that signal densities become much more similar. Average scan signals, which have a larger dynamical range and greater signal-to-noise ratio than the individual scans, can then be obtained. An implementation in \R of the above calibration method is made available for free.
Key words:
microarray scanner; bias; dark noise; offset; zero level; affine model; calibration; normalization; dynamic range; photomultiplier tube; digital-to-analogue converter; image analysis; intensity dependent effects; background subtraction