Dear All I have just seen a result which makes me either suspect the validity of print-tip loess normalisation, or wonder if there is a bug in bioconductor. I am performing print-tip loess normalisation on a dataset which I have already performed background correction on outside of bioconductor. Here is my code: > dataset[2896,1]@maRf chick1a_chick1.21406.bgc [1,] 6 > dataset[2896,1]@maRb chick1a_chick1.21406.bgc [1,] 0 > dataset[2896,1]@maGf chick1a_chick1.21406.bgc [1,] 228 > dataset[2896,1]@maGb chick1a_chick1.21406.bgc [1,] 0 > maM(dataset[2896,1]) chick1a_chick1.21406.bgc [1,] > ma <- maNorm(dataset, norm='printTipLoess') -5.247928 > ma[2896,1]@maM chick1a_chick1.21406.bgc [1,] 5.169925 As can be seen, my raw Cy5 value is 6 and the raw Cy3 value is 228. My raw ratio is 6/228 = 0.026, and my raw log2(ratio) is -5.2479. All is as expected. But how on earth has the normalisation method turned my normalised ratio to +5.169???? I clearly have a spot here which, if anything, is down-regulated in the Cy5 channel. Yet my normalised log ratio comes out at +5 - which suggests a 32-fold increase in the Cy5 channel, from data where cy5=6 and cy3=228. I find this result pretty hard to accept.... Thanks in advance for the help Mick

Hi Michael, One way to get a feel if the normalization is performing as you have expected is to look at the diagnostic plots before and after noramlization. You are focusing on one spot at the lower end of A values. Could you send me the corresponding MA-plot prior to normalization. This will provide an idea of the variability of your data around A=7. In addition, are there any "quality weights" information associated with this particular spot? Cheers Jean On Wed, 9 Jun 2004, michael watson (IAH-C) wrote: > Dear All > > I have just seen a result which makes me either suspect the validity of > print-tip loess normalisation, or wonder if there is a bug in > bioconductor. > > I am performing print-tip loess normalisation on a dataset which I have > already performed background correction on outside of bioconductor. > Here is my code: > > > dataset[2896,1]@maRf > chick1a_chick1.21406.bgc > [1,] 6 > > dataset[2896,1]@maRb > chick1a_chick1.21406.bgc > [1,] 0 > > dataset[2896,1]@maGf > chick1a_chick1.21406.bgc > [1,] 228 > > dataset[2896,1]@maGb > chick1a_chick1.21406.bgc > [1,] 0 > > maM(dataset[2896,1]) > chick1a_chick1.21406.bgc > [1,] > > ma <- maNorm(dataset, norm='printTipLoess') > -5.247928 > > ma[2896,1]@maM > chick1a_chick1.21406.bgc > [1,] 5.169925 > > > > As can be seen, my raw Cy5 value is 6 and the raw Cy3 value is 228. My > raw ratio is 6/228 = 0.026, and my raw log2(ratio) is -5.2479. All is > as expected. > > But how on earth has the normalisation method turned my normalised ratio > to +5.169???? I clearly have a spot here which, if anything, is > down-regulated in the Cy5 channel. Yet my normalised log ratio comes > out at +5 - which suggests a 32-fold increase in the Cy5 channel, from > data where cy5=6 and cy3=228. > > I find this result pretty hard to accept.... > > Thanks in advance for the help > > Mick > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor >