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

[Maciej Jończyk]

Thank you Loren, I'll ignore this error.

>From: Loren Engrav <engrav <at> u.washington.edu>
>Subject: Re: [MAANOVA] Warnings in fitmaanova & information request
>Newsgroups: gmane.science.biology.informatics.conductor
>Date: 2010-03-12 02:07:33 GMT (1 day, 14 hours and 34 minutes ago)
>
>Greetings
>
>If you are asking is this warning significant
>
>> Warning messages:
>1: In any(result$random[idx.mainterm]) :
>  coercing
>> argument of type 'double' to logical
>2
>
>I have used maanova many many times and see that message every time and
the
>maanova people indicated I should ignore it

But I have next problem and I want to ask for advice.

*
I pasted some of the previous supplied information (I can't respond from
list level,
I respond to verification message but it was a day ago and nothing
happens :( ).


#####################################################################
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).

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.

######################################################################

After "fitmaanova":


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


I wanted to set up contrasts for interaction terms:

dlc vs dlk
dhc vs dhk
dlc vs dhc
dlk vs dhk

I constructed design matrix (I suppose that variants are ordered
alphabetically).

>cont_matx=matrix(c(1,-1,0,0,0,0,1,-1,1,0,-1,0,0,1,0,-1),nrow=4,byrow=T)

And I tried to perform test


>test=matest(eth_logrg,model.full_nodye,term="linia:temp",Contrast=cont_matx,
n.perm=61,shuffle.method="resid")

But it gave error message:

Error: The number 1 test is not estimable

What is wrong? Am I included too many effects in fitmaanova?
I have four replications of the experiment, I think it is enought.
Or maybe my contrasts' specification is wrong?
I haven't a clue what is wrong.

Help will be very appreciated.

Best wishes,

Maciej


Maciej Jończyk PhD
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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