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Sebastian Mueller
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10
@sebastian-mueller-2753
Last seen 9.6 years ago
dear members,
I have a general question about the creation of design matrices for
non-reference designs in limma.
The set-up of the experiment I'm refering to looks like this:
------------------
0h 1h 2h 4h
3 7 11
A > C > E > G
^ 1 ^ 5 ^ 9 ^ 13
v 2 v 6 v 10 v 14
B > D > F > H
4 8 12
------------------
The experiment consists, as you hopefully can guess, of 14 arrays
(each arrow
is a two-color array which points from cyc5 to cyc3).
Array 1 and 2 (5-6,9-10,13-14) correspond to a dye-swaps.
Each letter (A-H) corresponds to another condition (however A and B
should be
the same condition)
So, I'm interrested in the contrasts A-B, C-D, E-F, G-H for the time
points
0h, 1h, 2h, 4h, respectively.
The easiest way to do this (from the limma guide), would probably be
to create
a target-frame...
targets <- as.data.frame(cbind(
Cy3=c("B","A","A","B","D","C","C","D","F","E","E","F","H","G"),
Cy5=c("A","B","C","D","C","D","E","F","E","F","G","H","G","H")))
rownames(targets) <- paste("Array",1:14)
> targets
Cy3 Cy5
Array 1 B A
Array 2 A B
Array 3 A C
Array 4 B D
Array 5 D C
Array 6 C D
Array 7 C E
Array 8 D F
Array 9 F E
Array 10 E F
Array 11 E G
Array 12 F H
Array 13 H G
Array 14 G H
... and creating a design-matrix:
> design=modelMatrix(targets,ref="A")
Found unique target names:
A B C D E F G H
> design
B C D E F G H
Array 1 -1 0 0 0 0 0 0
Array 2 1 0 0 0 0 0 0
Array 3 0 1 0 0 0 0 0
Array 4 -1 0 1 0 0 0 0
Array 5 0 1 -1 0 0 0 0
Array 6 0 -1 1 0 0 0 0
Array 7 0 -1 0 1 0 0 0
Array 8 0 0 -1 0 1 0 0
Array 9 0 0 0 1 -1 0 0
Array 10 0 0 0 -1 1 0 0
Array 11 0 0 0 -1 0 1 0
Array 12 0 0 0 0 -1 0 1
Array 13 0 0 0 0 0 1 -1
Array 14 0 0 0 0 0 -1 1
cont=makeContrasts(C-D,E-F,G-H,levels=design)
...
---------------------
As I actually don't have a reference design, I'm wondering if this is
ok to do
this.
My problem is that I can't get the contrast A-B from this design. I
already
tried to append a colum A to this matrix by hand, but calling
>lmFit(MA, design)
gives me: Coefficients not estimable: A
As I probably haven't really understood the concept of creating a
design
matrix (I have already looked trough the limma guide), I was wondering
if
someone knows a good tutorial or book (I was already looking for
hours, but
there is not such a thing for limma and time series analysis)
I'm espacially wondering how I can solve this problem intuitively (as
I was
going to use limma for my future time-series experiments) and I also
would
like to know if my approch to tackle this problem is appropriate in
general.
Thanks a lot
Sebastian Mueller
------
Max-Planck-Institute for Chemical Ecology
Department of Biochemistry
Hans-Knoell-Strasse 8
D-07745 Jena
Germany
