Entering edit mode
At 03:46 PM 24/12/2003, Simon Melov wrote:
>when use the following series of commands to plot non-normalized data
in
>an MA plot highlighting the buffer controls and all other genes, I
get a
>very strange error. My feeling is this is occurring during the
>normalization itself.
What you are describing seems impossible. limma does not do any
adjustment
of A-values anywhere unless you used normalizeBetweenArrays(). In
particular I don't understand how you can attribute errors to the
normalization step since your code and text indicate that you haven't
done
any normalization.
Perhaps you are plotting different things in GeneTraffic and limma.
Have
you background corrected in both? With the same background measure?
Have
you done any filtering of spots in GeneTraffic? Have you identified
the
same group of Buffer spots in both programs? If you use plotMA(RG)
where do
the Buffer spots appear?
Without seeing a reproducible example, it's not possible to say any
more
than this.
Gordon
>files <- dir(pattern="*.gpr")
>RG <- read.maimages(files,source="genepix")
>targets <- readTargets("targets.txt")
>RG$genes <- readGAL()
>RG$printer <- getLayout(RG$genes)
>targets <- readTargets("targets.txt")
>types <- readSpotTypes("Spottypes.txt")
>RG$genes$Status <- controlStatus(types, RG)
>
>Matching patterns for: ID Name
>Found 25088 gene
>Found 32 Calibration-1
>Found 32 Calibration-2
>Found 32 Calibration-3
>Found 32 Calibration-4
>Found 32 Calibration-5
>Found 32 Calibration-6
>Found 32 Calibration-7
>Found 32 Calibration-8
>Found 32 Calibration-9
>Found 32 Calibration-10
>Found 512 Empty
>Found 288 Buffer
>Found 0 No data
>Found 32 Human Cot
>Found 32 Mouse Cot
>Found 32 B Actin
>Found 32 Poly A
>Found 32 Salmon Sperm
>Setting attributes: values Color
>
>plotMA(RG,value=c("gene","Buffer"),col=c("black","blue"))
>
>In the input data I have checked for correct assignation of block,
column,
>row (I am using gpr files) and gene identity. After normalization my
>buffers (which are negative controls) are displayed across the range
of A
>values in the MA plot (as seen below). However, most of my buffers
have
>very low A values (if you calculate manually). Somehow, between
loading
>and calculation of the A values, limma is adjusting these buffer
values so
>that they are spread across the range. I have checked everything I
can
>(i.e. are the individual values (R, Rb etc) being pulled from the
correct
>columns in the gpr files, and being assigned to the correct position
in
>the matrix. They are.
>
>As a final check, I plotted the same data in Genetraffic, and then in
>limma (both non-normalized), with the buffers highlighted in both
plots.
>Its the same data, but you can see when the raw data is plotted in
limma
>versus in Genetraffic, it is somehow being plotted incorrectly. There
are
>288 buffer spots being highlighted in blue in each graph, but as you
can
>see, the values in the limma graph are all spread out. This seems to
be
>occurring during the normalization function, as the values going into
the
>normalization function are correct, but those coming out are wrong.
>Individually checking several of the buffer values confirms this. I
am not
>clear how this is occurring. Unless I am doing something very
>fundamentally wrong, then this is a big problem.
>
>Please help!
>
>thanks
>
>Simon.
>
>Blue are the buffer controls in both cases. 256 of the buffer values
are
>below an A value of 7. Only 8 values are above 8. But in the limma
plot,
>most buffers are being plotted with an A value of greater than 8,
which is
>clearly fundamentally different.
