[BioC] Loop design - biological, technical replication and contrasts
Gordon K Smyth
smyth at wehi.EDU.AU
Sat Mar 6 02:34:18 CET 2010
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 biol.uw.edu.pl
> To: <bioconductor at stat.math.ethz.ch>
> Cc: Maciej Jo?czyk <mjonczyk at biol.uw.edu.pl>
> 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
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