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Question: limma: print-tip loess and empty spots
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gravatar for Gordon Smyth
11.1 years ago by
Gordon Smyth33k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth33k wrote:
Dear Adrian, Raw M-values are always defined to be log(Red/Green), i.e., 635/532. limma always computes them like that, because that's the definition. If you think you need to change this, you're almost certainly not thinking about your data in the best possible way. Correct allowance for which treatments have been labelled red and green is done at a later stage, not at the normalization stage. limmaGUI provides plenty of facilities to do this, but if you export the data out of the limma to treat it using other tools, then obviously this becomes your responsibility. BTW, limmaGUI provides features for checking data quality, such as MA-plots and image plots. Best wishes Gordon >Date: Wed, 30 May 2007 15:27:33 -0700 >From: "Adrian Steward" <adrian.steward0405 at="" gmail.com=""> >Subject: Re: [BioC] limma: print-tip loess and empty spots >To: keith at wehi.edu.au, bioconductor at stat.math.ethz.ch > >Hi Keith et al., > >Thanks for replying, I've been trying to learn limma as I go. I haven't >gotten into the linear modeling portion, but am rather using limma to >pre-process (e.g. normalize) my data to evaluate its quality before I get >serious with limma GUI for contrasts, heat maps, and all the nice stuff it >can do. Thus far, I have found no way to change the default setting for >635/532 - regardless of how the data is read in, the ratios are calculated >this way by limma. > >Is there a bit of code I can enter into the command line to change this? >All suggestions are welcome. > >Thanks! > >AM
ADD COMMENTlink modified 11.1 years ago • written 11.1 years ago by Gordon Smyth33k
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gravatar for Adrian Steward
11.1 years ago by
Adrian Steward50 wrote:
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ADD COMMENTlink written 11.1 years ago by Adrian Steward50
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gravatar for Gordon Smyth
11.1 years ago by
Gordon Smyth33k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth33k wrote:
Dear Adrian, At 08:36 AM 2/06/2007, Adrian Steward wrote: >Thank you for your reply, Dr. Smyth. > >I do not yet completely understand exactly HOW normalizing works >(I've seen the data, transformations, and so I know what it does, >just not how, yet) but it appears to me that I can simply change the >sign of the normalized output to make the proper tests In general, you cannot simplify the constructions of tests by swapping the sign of the normalized log-ratios. The only experiment in which people might be tempted to do this is a simple replicated comparison using two-colour arrays with dye-swaps (and you have given no indication that this is your experiment.) For anything more complicated, swapping the signs of the log-ratios would only complicate matters. Even for the replicated comparison, swapping the log-ratios is unhelpful because it prevents the inclusion of probe-specific dye-effects in the model. > (or as someone else stated, reverse the contrast / estimate statements). >You picked up on my motivations here - I am chiefly concerned that >the exported normalized data has proper signs The normalized data already has what we consider to be the "proper" signs. > because at present I am required to do all of my linear modeling > in SAS, and large datasets need to be 'read in.' I personally > would rather do it all in R which is why I am running things in > parallel to make the case for limma-only analysis. You can certainly fit linear models in SAS, but you can't do a limma empirical Bayes analysis. >You people are both programmers and teachers, and thanks for your >patience with the noobs. > >AM You can easily change the signs of columns of data in either R or SAS. You could get advice on how to do this from the R help list. But don't expect this from me or Keith because I believe it is undesirable. There is absolutely no reason why linear modelling in SAS or R requires any prior fudging of the data. You can easily handle the data as it actually is. Spend a little more time understanding how linear modelling works for microarray data, then you'll see why this is so. That would be time much better spent than trying to persuade limmaGUI to do what it doesn't want to do. Best wishes Gordon
ADD COMMENTlink written 11.1 years ago by Gordon Smyth33k
Quoting Gordon Smyth <smyth at="" wehi.edu.au="">: > Dear Adrian, > > At 08:36 AM 2/06/2007, Adrian Steward wrote: >> Thank you for your reply, Dr. Smyth. >> >> I do not yet completely understand exactly HOW normalizing works >> (I've seen the data, transformations, and so I know what it does, >> just not how, yet) but it appears to me that I can simply change the >> sign of the normalized output to make the proper tests > > In general, you cannot simplify the constructions of tests by > swapping the sign of the normalized log-ratios. The only experiment > in which people might be tempted to do this is a simple replicated > comparison using two-colour arrays with dye-swaps (and you have given > no indication that this is your experiment.) For anything more > complicated, swapping the signs of the log-ratios would only > complicate matters. Even for the replicated comparison, swapping the > log-ratios is unhelpful because it prevents the inclusion of > probe-specific dye-effects in the model. > >> (or as someone else stated, reverse the contrast / estimate statements). >> You picked up on my motivations here - I am chiefly concerned that >> the exported normalized data has proper signs > > The normalized data already has what we consider to be the "proper" signs. > >> because at present I am required to do all of my linear modeling >> in SAS, and large datasets need to be 'read in.' I personally >> would rather do it all in R which is why I am running things in >> parallel to make the case for limma-only analysis. > > You can certainly fit linear models in SAS, but you can't do a limma > empirical Bayes analysis. > >> You people are both programmers and teachers, and thanks for your >> patience with the noobs. >> >> AM > > You can easily change the signs of columns of data in either R or > SAS. You could get advice on how to do this from the R help list. But > don't expect this from me or Keith because I believe it is undesirable. > > There is absolutely no reason why linear modelling in SAS or R > requires any prior fudging of the data. You can easily handle the > data as it actually is. Spend a little more time understanding how > linear modelling works for microarray data, then you'll see why this > is so. That would be time much better spent than trying to persuade > limmaGUI to do what it doesn't want to do. > > Best wishes > Gordon > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor Very reluctantly I will jump in, because I remember my own experience as a total "newbie" to this world not long ago... and I feel that the reason why Adrian is asking about changing the signs may have little to do with linear modelling and what it does to the data. At least, I had similar questions... but I don't want to insult Adrian by comparing him with me ;-) When I started with limma (actually limmaGUI), I did it with data from dye-swap experiments. After normalisation, the sign of the M values is determined by the log2 of the ratio Cy5/Cy3, as Gordon explained. That's the convention. Just like we generally agree to call Cy3 the Green channel, and Cy5 the Red channel... which was counterintuitive for somebody like me, who was used to using Cy3 in microscopy and it's usually seen as red (reddish, but the computer then goes and paints it bright red)... Just a convention. According to that convention, teh signs of my dye-swapped arrays were either positive or negative, depending on teh orientation of the hyb in question. At first that was a little disorientationg, because I had to make sure I remember which array was hyb in what order (info that's stored in the 'targets' object, if using limma). However, one doesn't need to worry about that. The normalised data (per array) I only look at it to check the quality of the hybs, really... to make a few MA plots and see general patterns, check for artifacts, etc. After that step, we take the normalised data, and we fit a linear model to it with the function 'lmFit'. Limma does this taking into account the orientation of the separate hybs (information present in the 'targets' object), and using a design matrix of our choice. Similarly if we want to specify particular contrasts. After this, we obtain M values that have the "correct" sign, according to whatever orientation we indicate in teh design matrix... so not only it's not necessary to change manually the signs of the normalised data, per array, but also, if we do so, we'd mess up the linear model fitting... which is the whole point about using Limma. So, if I have four slides, comparing samples A and B, with two dye swaps: Array Cy3 Cy5 1 A B 2 A B 3 B A 4 B A and I am ultimately interested in B-A, and I have a gene X that has higher expression in sample B than in A... when I normalise the data, the M values for that gene X will be positive in arrays 1 and 2, and negative in arrays 3 and 4 [log2(Cy5/Cy3)]. After fitting teh linear modelling, where we indicate we want the comparison B-A, what we'll get is a single M value, and its sign will be _positive_. I am not sure if this helped any, or it was too obvious to be of any use... I just felt you were using limma only half-way, stopping at the normalisation stage, and ignoring the 'best' part of it: the linear modelling. Jose -- Dr. Jose I. de las Heras Email: J.delasHeras at ed.ac.uk The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6513374 Institute for Cell & Molecular Biology Fax: +44 (0)131 6507360 Swann Building, Mayfield Road University of Edinburgh Edinburgh EH9 3JR UK
ADD REPLYlink written 11.1 years ago by J.delasHeras@ed.ac.uk1.9k
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