Hi everyone, I'm using AROMA's implementation of the CRMA v2 method to get copy number estimates for cancer samples, and I'm getting a very unusual result. Many of the samples have a chromosome where AROMA has called primarily copy number gains or losses, and the losses are mixed in with each other. That is, if you plot the probes' intensities by their positions on the chromosome, you see large stretches (~10,000 probes) where there are no intensities in the normal range (corresponding to no gain or loss), and there are intensities both above and below the normal range, mixed in with each other along the chromosome. It is as if the plots for a chromosome with a long deletion and a chromosome with a long addition were laid atop each other. It seems implausible that a cancer sample would have copy number gains and losses mixed in with each other in such small intervals, over such large stretches of chromosome, without any loci having the usual 2 copies, so I suspect the normalization or the affy array is the source of this phenomenon. I looked at the data without using AROMA, and the phenomenon was not evident. I re-normalized the data 3 times, each time using only one step of the AROMA normalization in isolation. The base position normalization step produced the phenomenon, and the allele crosstalk calibration and the fragment length normalization steps did not. Any thoughts on what I'm seeing and on how the base pair normalization could cause it would be very appreciated. Thanks, Patrick
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