[BioC] [MAANOVA] Warnings in fitmaanova & information request

Thu Mar 11 12:11:10 CET 2010

Firstly I want to apologise for my top-posting.
I won't do that in future.

Dear list members,

I'm trying to analyse simple loop-design in MAANOVA package.
I've used two-colour spotted oligo arrays.

I have two maize lines (linia): dl, dh;
two conditions (temp): c (cold), k (control).

Here is my design:

dlc ---- dlk
|    X    |
dhc ---- dhk

with two more hybridizations, dlc vs dhk and dlk vs dhc ("X" in this
scheme).

I repeated this scheme four times, with dye swap (two labelings in
both directions).

Technical replication is within each replicated scheme. So within
scheme all instances of each sample (samples: dlc, dlk, dhc, dhk)
is from the same isolation (i.e. in each loop each RNA is used
three times in different hybridizations).

Here is my design file (powt_b is a number of biological replicate):

Array		Dye	Sample	linia	temp	powt_b
dhk_dhc093	Cy5	1	dh	c	1
dhk_dhc093	Cy3	2	dh	k	1
dhc_dhk104	Cy5	3	dh	k	2
dhc_dhk104	Cy3	4	dh	c	2
dhk_dhc116	Cy5	5	dh	c	3
dhk_dhc116	Cy3	6	dh	k	3
dhc_dhk016	Cy5	7	dh	k	4
dhc_dhk016	Cy3	8	dh	c	4
dhk_dlk094	Cy5	9	dl	k	1
dhk_dlk094	Cy3	2	dh	k	1
dlk_dhk105	Cy5	3	dh	k	2
dlk_dhk105	Cy3	10	dl	k	2
dhk_dlk117	Cy5	11	dl	k	3
dhk_dlk117	Cy3	6	dh	k	3
dlk_dhk139	Cy5	7	dh	k	4
dlk_dhk139	Cy3	12	dl	k	4
dlc_dlk092	Cy5	9	dl	k	1
dlc_dlk092	Cy3	13	dl	c	1
dlk_dlc106	Cy5	14	dl	c	2
dlk_dlc106	Cy3	10	dl	k	2
dlc_dlk118	Cy5	11	dl	k	3
dlc_dlk118	Cy3	15	dl	c	3
dlk_dlc023	Cy5	16	dl	c	4
dlk_dlc023	Cy3	12	dl	k	4
dlc_dhc095	Cy5	1	dh	c	1
dlc_dhc095	Cy3	13	dl	c	1
dhc_dlc107	Cy5	14	dl	c	2
dhc_dlc107	Cy3	4	dh	c	2
dlc_dhc119	Cy5	5	dh	c	3
dlc_dhc119	Cy3	15	dl	c	3
dhc_dlc136	Cy5	16	dl	c	4
dhc_dlc136	Cy3	8	dh	c	4
dlk_dhc101	Cy5	1	dh	c	1
dlk_dhc101	Cy3	9	dl	k	1
dhc_dlk103	Cy5	10	dl	k	2
dhc_dlk103	Cy3	4	dh	c	2
dlk_dhc121	Cy5	5	dh	c	3
dlk_dhc121	Cy3	11	dl	k	3
dhc_dlk015	Cy5	12	dl	k	4
dhc_dlk015	Cy3	8	dh	c	4
dhk_dlc100	Cy5	13	dl	c	1
dhk_dlc100	Cy3	2	dh	k	1
dlc_dhk102	Cy5	3	dh	k	2
dlc_dhk102	Cy3	14	dl	c	2
dhk_dlc120	Cy5	15	dl	c	3
dhk_dlc120	Cy3	6	dh	k	3
dlc_dhk140	Cy5	7	dh	k	4
dlc_dhk140	Cy3	16	dl	c	4

As required, Sample denotes distinct RNAs. So, e.g. condition
"dl c" has the same number within single replication of entire
scheme (what makes three technical replicates of RNA withnin
biological replicate).

My data file is organised exactly as described in manual.

I've read the data:
>eth_logrg=read.madata("./eth4maan",designfile=".des4maanova",
arrayType="twoColor",header=TRUE,spotflag=FALSE,log.trans=TRUE,
metarow=2,metacol=3,row=4,col=5,probeid=1,intensity=6)

I wrote the model. I want to include factors linia, powt and
their interaction as a fixed effects. I also included Sample,
powt_b and Array as a random effects. In this way I want to
include effect of techical replication. I didn't include Dye
effect, because the experiment was dye balanced, and data
normalized.

>model.full_nodye=fitmaanova(eth_logrg,formula=~Array+Sample
+powt_b+linia+temp+linia:temp,random=~Array+Sample+powt_b)

After this code I get warning messages. Here it is:

...
Finish gene number 42500 ...
Finish gene number 42600 ...
Finish gene number 42700 ...
Finish gene number 42800 ...
Finish gene number 42900 ...
Finish gene number 43000 ...
Finish gene number 43100 ...
Finish gene number 43200 ...
Finish gene number 43300 ...
Warning messages:
1: In any(result$random[idx.mainterm]) :
  coercing argument of type 'double' to logical
2: In any(parsed.formula$random) :
  coercing argument of type 'double' to logical
3: In any(result$random[idx.mainterm]) :
  coercing argument of type 'double' to logical
4: In any(parsed.formula$random) :
  coercing argument of type 'double' to logical

What it means? I've got the object I specified, is it valid?

> summary(model.full_nodye)
                 Length  Class   Mode
probeid            43381 -none-  character
yhat             2082288 -none-  numeric
S2                173524 -none-  numeric
loops              43381 -none-  numeric
S2.level               3 -none-  character
G                  43381 -none-  numeric
Array            1041144 -none-  numeric
Array.level           24 -none-  character
Sample            694096 -none-  numeric
Sample.level          16 -none-  numeric
powt_b            173524 -none-  numeric
powt_b.level           4 -none-  numeric
linia              86762 -none-  numeric
linia.level            2 -none-  character
temp               86762 -none-  numeric
temp.level             2 -none-  character
linia:temp        173524 -none-  numeric
linia:temp.level       4 -none-  character
model                 12 mamodel list
subCol                 1 -none-  logical

I also would like to request more information about usage of
the MAANOVA package.
I've read manual and material from this list and
http://churchill.jax.org/software/rmaanova.shtml.

In particular I'd like to find out how to set up contrasts,
like e.g. dhc vs dhk.

Best Regards,

Maciej Jończyk
Department of Plant Molecular Ecophysiology
Institute of Plant Experimental Biology
Faculty of Biology, University of Warsaw
02-096 Warszawa, Miecznikowa 1

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