Although you don't say exactly, I assume that your question is why the two results MA.1 and MA.2 are slightly different. Zero weights in the loess normalization are almost but not quite the same as NA. Observations which are NA really are ignored entirely. Observations which have zero weight are given no weight in the local regressions, but are used by the loess function to determine the span neighborhood of each local point. Hence zero weights in loess are nearly but not quite the same as missing observations. This behavior is a characteristic of the loess() function which is used by normalizeWithinArrays(). The effect of zero weights is usually close enough to treating the value as missing, unless the propotion of zero weights is exceptionally high. The difference is seldom important in practice, and doesn't seem important in your case. Best wishes Gordon > Date: Mon, 20 Feb 2006 12:58:15 +0100 > From: Ana Conesa <aconesa at="" ivia.es=""> > Subject: [BioC] Limma: bad spots flagged out? > To: bioconductor at stat.math.ethz.ch > Message-ID: <7.0.0.16.0.20060220124132.02020ff0 at ivia.es> > Content-Type: text/plain; charset="iso-8859-1" > > > Dear list, > I have a doubt about the real use of spots weights during > normalization in limma. According with the documentation spots weights > equal to 0 are ignored during normalization (and other posterior > analyses) , and although these spots are not removed they do not have > any influence on the rest. I had observed some strange behavior on my > normalized data and made a tried to make a check on this. What I did > was to replace weights==0 spots by NA and redo analysis. I have > found the results do no spots are adequately ignored by limma or I made a conceptual > mistake > in this check. This is exactly the code I used for checking: > > RG.b <- backgroundCorrect(RG) > > MA.1 <- normalizeWithinArrays(RG.b) > > RG.b$G[RG.b$weights==0] <- NA > > RG.b$R[RG.b$weights==0] <- NA > > MA.2 <- normalizeWithinArrays(RG.b) > > MA.1$M[2,] > BFN33 S50.05 S65.02 S65.03 S71.02 > S75.01 S77.03 control control control control > -0.96794887 0.06715693 -0.08766477 -0.50161127 -1.25216169 > -0.80724650 -0.61351625 -0.80751427 -0.49960303 -0.66912129 > 0.27447918 > > MA.2$M[2,] > BFN33 S50.05 S65.02 S65.03 S71.02 > S75.01 S77.03 control control control control > -0.94108221 0.04803980 -0.08030398 -0.49226094 NA > -0.79588160 NA -0.77182854 NA -0.61426784 > 0.18076174 > Thank you > Ana > > O @@@@@ Ana Conesa, PhD. > @@@ O @@ O @ Centro de Gen?mica > @ O @@@@ O @ Instituto Valenciano de Investigaciones Agrarias > (IVIA) > @@@ O @@@@ @@@@ O @ 46113 Moncada (Valencia) SPAIN > || Tel. +34 963424000 ext.70161; Fax. +34 963424001 > || email: aconesa at ivia.es