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 Jo?czyk Department of Plant Molecular Ecophysiology Institute of Plant Experimental Biology Faculty of Biology, University of Warsaw 02-096 Warszawa, Miecznikowa 1 ___________________________________ NOCC, http://nocc.sourceforge.net -- This email was Anti Virus checked by Astaro Security Gateway. http://www.astaro.com

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 > From: Maciej Jo?czyk <mjonczyk at="" biol.uw.edu.pl=""> > Reply-To: Maciej Jo?czyk <mjonczyk at="" biol.uw.edu.pl=""> > Date: Thu, 11 Mar 2010 12:11:10 +0100 > To: "bioconductor at stat.math.ethz.ch" <bioconductor at="" stat.math.ethz.ch=""> > Cc: Maciej Jo?czyk <mjonczyk at="" biol.uw.edu.pl=""> > Subject: [BioC] [MAANOVA] Warnings in fitmaanova & information request > > 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_d > hc093 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_dlk0 > 92 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_dhc1 > 21 Cy5 5 dh c 3 dlk_dhc121 Cy3 11 dl k 3 dhc_dlk015 Cy5 12 dl k 4 dhc_dlk015 C > y3 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 Jo?czyk Department of Plant Molecular Ecophysiology Institute > of Plant Experimental Biology Faculty of Biology, University of Warsaw 02-096 > Warszawa, Miecznikowa 1 ___________________________________ NOCC, > http://nocc.sourceforge.net -- This email was Anti Virus checked by > Astaro Security Gateway. > http://www.astaro.com _______________________________________________ Biocond > uctor mailing > list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioc > onductor Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor

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+li nia+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=con t_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 Jo?czyk PhD Department of Plant Molecular Ecophysiology Institute of Plant Experimental Biology Faculty of Biology, University of Warsaw 02-096 Warszawa, Miecznikowa 1 ___________________________________ NOCC, http://nocc.sourceforge.net -- This email was Anti Virus checked by Astaro Security Gateway. http://www.astaro.com