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




On 5/23/07, Keith Satterley <keith@wehi.edu.au > wrote:
>
> Hi Adrian
>
> When you are using limmaGUI you can specify a set of parameters under the
> Linear
> Model Menu, either as A-B or B-A, which reverses the Fold Change sign in
> the
> TopTable. For example, My test Targets file is:
> SlideNumber     FileName        Cy3     Cy5     Date
> 1               slide1.gpr      B       A       11/07/2004
> 2               slide2.gpr      B       A       11/07/2004
> 3               slide3.gpr      B       A       11/07/2004
> 4               slide4.gpr      B       A       11/07/2004
> 5               slide5.gpr      B       A       11/07/2004
> 6               slide6.gpr      B       A       11/07/2004
>
> If my parameterisation is B minus A, The first two lines in my TopTable
> for the
> relevant columns are:
> "Name"          "logFC"         "AveExpr"   "t"
> "AA668821"      "-5.931"        "11.36"     "-52.24"
> "H87471"        "5.258"         "10.52"     "40.8"
> whereas if my parameterisation is A minus B,  The first two lines in my
> TopTable
> for the relevant columns are:
> "Name"          "logFC"         "AveExpr"   "t"
> AA668821"       "5.931"         "11.36"     "52.24"
> "H87471"        "- 5.258"        "10.52"     "-40.8
>
> The LogFC values are reversed in sign.
>
> Is this helpful, or is your problem something different?
>
> I'm using R2.5.0, limma 2.10.4 and affylmGUI 1.12.0 on Windows for this
> test
> problem,
>
> cheers,
>
> Keith Satterley
> ===================
> Maintainer:limmaGUI
> Bioinformatics Division
> The Walter and Eliza Hall Institute of Medical Research
> Parkville, Melbourne,
> Victoria, Australia
> =======================
>
> Adrian Steward wrote:
> > Thank you Dr. Smyth.
> >
> > Having processed some raw data in limma, I am seeing something
> peculiar.  I
> > used Cy5 as reference and Cy3 as target, on the Axon platform using Gene
> Pix
> > Pro (latest version) to produce genepix results files.
> >
> > Using Limma GUI, regardless of what I set the 'targets' file to
> indicate, my
> > raw log ratio data output always come out from limma as the opposite
> sign
> > than it should be; in other words, what should be negative is positive,
> and
> > vice versa.  I know I set the calculation 532/635 correctly in Genepix.
> >
> > Is there something that I am missing?
> >
> > Thanks,
> >
> > Adrian
> >
> > On 5/22/07, Gordon K Smyth <smyth@wehi.edu.au> wrote:
> >> Dear Adrian,
> >>
> >> I assume that you're already read the limma User's Guide advice:
> >>
> >> "Print-tip loess is also unreliable for small arrays with less than,
> say,
> >> 150 spots per print-tip
> >> group. Even larger arrays may have particular print-tip groups which
> are
> >> too small for printtip
> >> loess normalization if the number of spots with non-missing M-values is
> >> small for one or more of
> >> the print-tip groups. In these cases one should either use global
> "loess"
> >> normalization or else
> >> use robust spline normalization"
> >>
> >> There are however special considerations for multispecies arrays, see
> >>
> >> Gilad, Y., Oshlack, A., Smyth, G. K., Speed, T. P., and White, K. P.
> >> (2006). Expression profiling
> >> in primates reveals a rapid evolution of human transcription factors.
> >> Nature 440, 242-245.
> >>
> >> Oshlack, A., Smyth, G. K., and Gilad, Y. (2007). Using DNA microarrays
> to
> >> study gene expression in
> >> closely related species. Bioinformatics. (Published online 23 March
> 2007).
> >>
> >> and perhaps
> >>
> >> Oshlack, A., Emslie, D., Corcoran, L., and Smyth, G. K. (2007).
> >> Normalization of boutique
> >> two-color microarrays with a high proportion of differentially
> expressed
> >> probes. Genome Biology 8,
> >> R2.
> >>
> >> Best wishes
> >> Gordon
> >>
> >> ------------- original message ----------------
> >> Adrian Steward adrian.steward0405 at gmail.com
> >> Mon May 21 19:52:56 CEST 2007
> >>
> >> Hi all,
> >>
> >> I am using the limma package to analyze a multi-species cDNA array,
> >> 2-colour
> >> reference design.  The problem is that because it is a multi-species
> (and
> >> tissue) array, and I am querying only 1 tissue, only 1/3 of the 15,000
> >> spots
> >> appear to correspond to cDNA in my samples, and the number of spots
> that
> >> actually get tested is around 3,500.  These spots are rather randomly
> >> located across the slides because of many libraries used in
> construction
> >> of
> >> the array.
> >>
> >> Before I get too far into my analysis, I read in the limma guide that
> >> print-tip loess within-slide normalization is not always a good choice
> for
> >> data with 'small' print tip groups.  I am assuming that a global loess
> >> normalization is the more appropriate approach in my case.
> >>
> >> Is my assumption reasonable?
> >>
> >> With thanks
> >>
> >> Adrian M.
> >>
> >>
> >> PS - I'm running R 2.3.1, limma 2.7.3, and limma GUI version 1.8.1
> >>
> >>
> >
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