Dear dr Smith and list members, lines l and h are biologically independent. I think there are three levels of variability: line (l and h); temperature (c and k) and isolation. I've thought that I can include line and temperature as a fixed effects and treat isolation as random effect. I can figure out that I should get into account also array effect. My main pursues are to get contrasts as I specified and account for technical replication (isolation). I think that I should include all of this effects. I'll try to use maanova package. Thank you, Best regards, Maciej PS. Are my previous codes for single channel analysis makes any sense? If I want to include line and temperature effects only would it be correct? I just want to make sure, so please write if it is completely wrong. From: Gordon K Smyth <smyth@...> Subject: Re: Loop design - biological, technical replication Newsgroups: gmane.science.biology.informatics.conductor Date: 2010-03-07 07:03:29 GMT (1 day, 1 hour and 46 minutes ago) Dear Maciej, You don't say, but I assume that lines l and h must be biologically independent. If this is true, then your experiment has 3 levels of variability: line, isolation and array. limma can only handle 2 levels. You could analyse data this in limma, treating biol_rep as a blocking variable, as in Section 8.2 of the User's Guide, but that would ignore the fact that h and l are biologically separate. Otherwise, try the maanova package. Best wishes Gordon ----------------------------------------------- Associate Professor Gordon K Smyth, NHMRC Senior Research Fellow, Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Vic 3052, Australia. Fax (03) 9347 0852, smyth at ... http://www.wehi.edu.au http://www.statsci.org/smyth On Sat, 6 Mar 2010, Maciej Jo?czyk wrote: > Dear dr Smith and list Members, > > I use single-channel analysis to deal with loop-design as suggested by > Naomi in some of her posts. > > Here is my targets with number of biological replication (replication of > entire scheme). Technical replication is within each replicated scheme. > So within scheme all instances of each sample (samples: lc, lk, hc, hk) > is from the same isolation (i.e. in each loop each RNA is used three > times in different hybridizations). > > So in my case technical replication include RNA samples, not > hybridizations. > > lines: l, h > temperaures: c(cold), k(control) > That two are concatenated in target file. > > Scheme is as follows: > > lc ---- lk > | | > hc ---- hk > > with two more hybridizations, lc vs hk and lk vs hc. > > This is my targets in original two-channel form (biol_rep states for > biological replication): > > SlideNumber FileName Cy3 Cy5 biol_rep > 93 c_093_DH_K_vs_DH_CHex.gpr hk hc 1 > 104 c_104_DH_CH_vs_DH_Kex.gpr hc hk 2 > 116 c_116_DHK_vs_DHCHex.gpr hk hc 3 > 16 c_016_DH_C_vs_DH_Kex.gpr hc hk 4 > 94 c_094_DH_K_vs_DL_Kex.gpr hk lk 1 > 105 c_105_DL_K_vs_DH_Kex.gpr lk hk 2 > 117 c_117_DHK_vs_DLKex.gpr hk lk 3 > 139 c_139_DL_K_vs_DH_Kex.gpr lk hk 4 > 92 c_092_DL_CH_vs_DL_Kex.gpr lc lk 1 > 106 c_106_DL_K_vs_DL_CHex.gpr lk lc 2 > 118 c_118_DLCH_vs_DLKex.gpr lc lk 3 > 23 c_023_DL_K_vs_DL_Cex.gpr lk lc 4 > 95 c_095_DL_CH_vs_DH_CHex.gpr lc hc 1 > 107 c_107_DH_CH_vs_DL_CHex.gpr hc lc 2 > 119 c_119_DLCH_vs_DHCHex.gpr lc hc 3 > 136 c_136_DH_C_vs_DL_Cex.gpr hc lc 4 > 101 c_101_DL_K_vs_DH_CHex.gpr lk hc 1 > 103 c_103_DH_CH_vs_DL_Kex.gpr hc lk 2 > 121 c_121_DLK_vs_DHCHex.gpr lk hc 3 > 15 c_015_DH_C_vs_DL_Kex.gpr hc lk 4 > 100 c_100_DH_K_vs_DL_CHex.gpr hk lc 1 > 102 c_102_DL_CH_vs_DH_Kex.gpr lc hk 2 > 120 c_120_DHK_vs_DLCHex.gpr hk lc 3 > 140 c_140_DL_C_vs_DH_Kex.gpr lc hk 4 > > I hope it clarifies my design. > > Best regards, > > > Gordon K Smyth <smyth at="" ...=""> nadawca : > >> Dear Maciej, >> >> I haven't been able to figure out from this or your previous post >> exactly what >> your experimental design is. I suspect this is why you haven't got >> replies >> yet. I can see that you have technical replicates, but I'm not quite >> clear >> that there is any biological replication. It's also unclear to me why >> you need >> a single channel analysis. Perhaps you could explain your design a >> bit more >> explicitly, including a data.frame with separate factors indicating >> temperature, maize line, biological rep, and any other factors you >> need to take >> into account? >> >> Best wishes >> Gordon >> >>> Date: Tue, 02 Mar 2010 13:01:23 +0100 >>> From: mjonczyk at ... >>> To: <bioconductor at="" ...=""> >>> Cc: Maciej Jo?czyk <mjonczyk at="" ...=""> >>> Subject: Re: [BioC] Loop design - biological, technical replication >>> and contrasts >>>> >>> Hi again, >>>> >>> I apologise for replying to my own post, but it helps keep track if >>> someone will be interested. >>>> >>> I analysed my data with single channel analysis in limma, according >>> to >>> Chapter 9. of limma usersguide. >>>> >>> I changed my targets file (to make it more condensed) and removed >>> suffix >>> which >>> identified biological replication. So my targets looks like: >>>> >>>> nt_trg >>> >>> SlideNumber FileName Cy3 Cy5 >>> 1 93 c_093_DH_K_vs_DH_CHex.gpr hk hc >>> 2 104 c_104_DH_CH_vs_DH_Kex.gpr hc hk >>> 3 116 c_116_DHK_vs_DHCHex.gpr hk hc >>> 4 16 c_016_DH_C_vs_DH_Kex.gpr hc hk >>> 5 94 c_094_DH_K_vs_DL_Kex.gpr hk lk >>> 6 105 c_105_DL_K_vs_DH_Kex.gpr lk hk >>> 7 117 c_117_DHK_vs_DLKex.gpr hk lk >>> 8 139 c_139_DL_K_vs_DH_Kex.gpr lk hk >>> 9 92 c_092_DL_CH_vs_DL_Kex.gpr lc lk >>> 10 106 c_106_DL_K_vs_DL_CHex.gpr lk lc >>> 11 118 c_118_DLCH_vs_DLKex.gpr lc lk >>> 12 23 c_023_DL_K_vs_DL_Cex.gpr lk lc >>> 13 95 c_095_DL_CH_vs_DH_CHex.gpr lc hc >>> 14 107 c_107_DH_CH_vs_DL_CHex.gpr hc lc >>> 15 119 c_119_DLCH_vs_DHCHex.gpr lc hc >>> 16 136 c_136_DH_C_vs_DL_Cex.gpr hc lc >>> 17 101 c_101_DL_K_vs_DH_CHex.gpr lk hc >>> 18 103 c_103_DH_CH_vs_DL_Kex.gpr hc lk >>> 19 121 c_121_DLK_vs_DHCHex.gpr lk hc >>> 20 15 c_015_DH_C_vs_DL_Kex.gpr hc lk >>> 21 100 c_100_DH_K_vs_DL_CHex.gpr hk lc >>> 22 102 c_102_DL_CH_vs_DH_Kex.gpr lc hk >>> 23 120 c_120_DHK_vs_DLCHex.gpr hk lc >>> 24 140 c_140_DL_C_vs_DH_Kex.gpr lc hk >>>> >>>> >>> I transform it to apropriate form: >>>> tgr_sc=targetsA2C(nt_trg) >>>> tgr_sc >>> >>> channel.col SlideNumber FileName Target >>> 1.1 1 93 c_093_DH_K_vs_DH_CHex.gpr hk >>> 1.2 2 93 c_093_DH_K_vs_DH_CHex.gpr hc >>> 2.1 1 104 c_104_DH_CH_vs_DH_Kex.gpr hc >>> 2.2 2 104 c_104_DH_CH_vs_DH_Kex.gpr hk >>> 3.1 1 116 c_116_DHK_vs_DHCHex.gpr hk >>> 3.2 2 116 c_116_DHK_vs_DHCHex.gpr hc >>> 4.1 1 16 c_016_DH_C_vs_DH_Kex.gpr hc >>> 4.2 2 16 c_016_DH_C_vs_DH_Kex.gpr hk >>> 5.1 1 94 c_094_DH_K_vs_DL_Kex.gpr hk >>> 5.2 2 94 c_094_DH_K_vs_DL_Kex.gpr lk >>> 6.1 1 105 c_105_DL_K_vs_DH_Kex.gpr lk >>> 6.2 2 105 c_105_DL_K_vs_DH_Kex.gpr hk >>> 7.1 1 117 c_117_DHK_vs_DLKex.gpr hk >>> 7.2 2 117 c_117_DHK_vs_DLKex.gpr lk >>> 8.1 1 139 c_139_DL_K_vs_DH_Kex.gpr lk >>> 8.2 2 139 c_139_DL_K_vs_DH_Kex.gpr hk >>> 9.1 1 92 c_092_DL_CH_vs_DL_Kex.gpr lc >>> 9.2 2 92 c_092_DL_CH_vs_DL_Kex.gpr lk >>> 10.1 1 106 c_106_DL_K_vs_DL_CHex.gpr lk >>> 10.2 2 106 c_106_DL_K_vs_DL_CHex.gpr lc >>> 11.1 1 118 c_118_DLCH_vs_DLKex.gpr lc >>> 11.2 2 118 c_118_DLCH_vs_DLKex.gpr lk >>> 12.1 1 23 c_023_DL_K_vs_DL_Cex.gpr lk >>> 12.2 2 23 c_023_DL_K_vs_DL_Cex.gpr lc >>> 13.1 1 95 c_095_DL_CH_vs_DH_CHex.gpr lc >>> 13.2 2 95 c_095_DL_CH_vs_DH_CHex.gpr hc >>> 14.1 1 107 c_107_DH_CH_vs_DL_CHex.gpr hc >>> 14.2 2 107 c_107_DH_CH_vs_DL_CHex.gpr lc >>> 15.1 1 119 c_119_DLCH_vs_DHCHex.gpr lc >>> 15.2 2 119 c_119_DLCH_vs_DHCHex.gpr hc >>> 16.1 1 136 c_136_DH_C_vs_DL_Cex.gpr hc >>> 16.2 2 136 c_136_DH_C_vs_DL_Cex.gpr lc >>> 17.1 1 101 c_101_DL_K_vs_DH_CHex.gpr lk >>> 17.2 2 101 c_101_DL_K_vs_DH_CHex.gpr hc >>> 18.1 1 103 c_103_DH_CH_vs_DL_Kex.gpr hc >>> 18.2 2 103 c_103_DH_CH_vs_DL_Kex.gpr lk >>> 19.1 1 121 c_121_DLK_vs_DHCHex.gpr lk >>> 19.2 2 121 c_121_DLK_vs_DHCHex.gpr hc >>> 20.1 1 15 c_015_DH_C_vs_DL_Kex.gpr hc >>> 20.2 2 15 c_015_DH_C_vs_DL_Kex.gpr lk >>> 21.1 1 100 c_100_DH_K_vs_DL_CHex.gpr hk >>> 21.2 2 100 c_100_DH_K_vs_DL_CHex.gpr lc >>> 22.1 1 102 c_102_DL_CH_vs_DH_Kex.gpr lc >>> 22.2 2 102 c_102_DL_CH_vs_DH_Kex.gpr hk >>> 23.1 1 120 c_120_DHK_vs_DLCHex.gpr hk >>> 23.2 2 120 c_120_DHK_vs_DLCHex.gpr lc >>> 24.1 1 140 c_140_DL_C_vs_DH_Kex.gpr lc >>> 24.2 2 140 c_140_DL_C_vs_DH_Kex.gpr hk >>>> >>> Next, I made design matrix >>>> >>>> u=unique(tgr_sc$Target) >>>> f=factor(tgr_sc$Target,levels=u) >>>> design=model.matrix(~0+f) >>>> colnames(design)=u >>>> design >>> >>> hk hc lk lc >>> 1 1 0 0 0 >>> 2 0 1 0 0 >>> 3 0 1 0 0 >>> 4 1 0 0 0 >>> 5 1 0 0 0 >>> 6 0 1 0 0 >>> 7 0 1 0 0 >>> 8 1 0 0 0 >>> 9 1 0 0 0 >>> 10 0 0 1 0 >>> 11 0 0 1 0 >>> 12 1 0 0 0 >>> 13 1 0 0 0 >>> 14 0 0 1 0 >>> 15 0 0 1 0 >>> 16 1 0 0 0 >>> 17 0 0 0 1 >>> 18 0 0 1 0 >>> 19 0 0 1 0 >>> 20 0 0 0 1 >>> 21 0 0 0 1 >>> 22 0 0 1 0 >>> 23 0 0 1 0 >>> 24 0 0 0 1 >>> 25 0 0 0 1 >>> 26 0 1 0 0 >>> 27 0 1 0 0 >>> 28 0 0 0 1 >>> 29 0 0 0 1 >>> 30 0 1 0 0 >>> 31 0 1 0 0 >>> 32 0 0 0 1 >>> 33 0 0 1 0 >>> 34 0 1 0 0 >>> 35 0 1 0 0 >>> 36 0 0 1 0 >>> 37 0 0 1 0 >>> 38 0 1 0 0 >>> 39 0 1 0 0 >>> 40 0 0 1 0 >>> 41 1 0 0 0 >>> 42 0 0 0 1 >>> 43 0 0 0 1 >>> 44 1 0 0 0 >>> 45 1 0 0 0 >>> 46 0 0 0 1 >>> 47 0 0 0 1 >>> 48 1 0 0 0 >>> attr(,"assign") >>> [1] 1 1 1 1 >>> attr(,"contrasts") >>> attr(,"contrasts")$f >>> [1] "contr.treatment" >>>> >>> *Is it correct form my design? I see, that it simply identifies what >>> RNA >>> was hybridized on each array. >>>> >>>> corfit=intraspotCorrelation(nt_img_lA,design) >>>> corfit$consensus >>> [1] 0.7341876 >>>> fit=lmscFit(nt_img_lAq,design,correlation=corfit$consensus) >>>> >>> I want to get contrasts "hc - hk", "lc - lk", "hc - lc", "hk - lk" >>> and also test effect of line and temperature. To do that I write >>> this >>> command: >>>> >>>> >>> >>> >> contr.matrix=makeContrasts(hc-hk,lc-lk,hc-lc,hk-lk,linia=(hc+hk-lc- lk)/2,temp=(hc+lc-hk-lk)/2,inter=(hc-lc)-(hk-lk),levels=design) >>>> >>> * I'm not 100% sure that it's correct. >>>> >>>> contr.fit=contrasts.fit(fit,contr.matrix) >>>> contr.fit=eBayes(contr.fit) >>>> >>> >>> >> wynik=decideTests(contr.fit,method="global",adjust.method="BH",p.value =0.05) >>>> summary(wynik) >>> hc - hk lc - lk hc - lc hk - lk linia temp inter >>> -1 5865 5039 3014 2685 3931 7382 1113 >>> 0 30922 33433 37177 38480 35896 28364 40776 >>> 1 6594 4909 3190 2216 3554 7635 1492 >>>> >>> From that it seem that there is a lot of differentially expressed >>> genes. >>> I feel that it isn't optimal design, here technical and biological >>> replications >>> are treated in the same manner, aren't they? >>>> >>> I've read about "duplicateCorrelation" command, is it possible to >>> combine it with single channel analysis? >>> Or I should rewrite target file (add number of replication) and >>> rewrite >>> contrasts >>> (e.g. hc-hk change to "((hc1+hc2+hc3+hc4)-(hk1+hk2+hk3+hk4))/4 >>> )? >>>> >>> And if I want to include a dye effect, I should only add column with >>> 1's >>> to my design, right? >>>> >>> Thank you for reading of my post. >>> I'd be very grateful for help. I've tried to analyse this data for a >>> along time >>> and I think limma is the best choice. >>>> >>> Yours sincerely, >>>> >>> Maciej Jo?czyk >>>> >>> Maciej Jo?czyk >>> Department of Plant Molecular Ecophysiology >>> Institute of Plant Experimental Biology >>> Faculty of Biology, University of Warsaw >>> 02-096 Warszawa, Miecznikowa 1 >> >> ______________________________________________________________________ >> The information in this email is confidential and intended solely for >> the addressee. >> You must not disclose, forward, print or use it without the permission >> of the sender. >> ______________________________________________________________________ >> >> -- >> This message has been 'sanitized'. This means that potentially >> dangerous content has been rewritten or removed. The following >> log describes which actions were taken. >> >> [ score: 10 ] >> 00000 Split unusually long Date: header. >> >> Anomy 0.0.0 : sanitizer.pl >> $Id: Sanitizer.pm,v 1.17 2001/08/07 15:16:46 bre Exp $ >> > > > 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 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