EdgeR design question?
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joseph ▴ 330
@joseph-1270
Last seen 10.2 years ago
Hi I am using edgeR to analyse an RNA-seq experiment with two factors, age (Young, Old) and genotype (Wt, Mutant). > targets            Age Genotype Sample1  Young       Wt Sample2  Young       Wt Sample3  Young       Wt Sample4  Young   Mutant Sample5  Young   Mutant Sample6  Young   Mutant Sample7    Old       Wt Sample8    Old       Wt Sample9    Old       Wt Sample10   Old   Mutant Sample11   Old   Mutant Sample12   Old   Mutant I know from previous experiments that the age effect is different between Wt and Mutant genotypes. I would like to find: 1. genes that change with age in Wt. 2. genes that change with age in Mutant. 3. genes that change with age differently between the genotypes (interaction). Can the following approach answer these questions? targets$Age <- relevel(targets$Age, ref="Young") targets$Genotype <- relevel(targets$Genotype, ref="Wt") design1 <- model.matrix(~Age + Age:Genotype, data=targets) > design1          (Intercept) AgeOld AgeYoung:GenotypeMutant AgeOld:GenotypeMutant Sample1            1      0                       0 0 Sample2            1      0                       0 0 Sample3            1      0                       0 0 Sample4            1      0                       1 0 Sample5            1      0                       1 0 Sample6            1      0                       1 0 Sample7            1      1                       0 0 Sample8            1      1                       0 0 Sample9            1      1                       0 0 Sample10           1      1                       0 1 Sample11           1      1                       0 1 Sample12           1      1                       0 1 attr(,"assign") [1] 0 1 2 2 attr(,"contrasts") attr(,"contrasts")$Age [1] "contr.treatment" attr(,"contrasts")$Genotype [1] "contr.treatment" I think coef=4 in design1 represents the genes that change with age in Mutant; but I don't know what coefficient represents the genes that change with age in Wt. design2 <- model.matrix(~Age*Genotype, data=targets) > design2          (Intercept) AgeOld GenotypeMutant AgeOld:GenotypeMutant Sample1            1      0              0                     0 Sample2            1      0              0                     0 Sample3            1      0              0                     0 Sample4            1      0              1                     0 Sample5            1      0              1                     0 Sample6            1      0              1                     0 Sample7            1      1              0                     0 Sample8            1      1              0                     0 Sample9            1      1              0                     0 Sample10           1      1              1                     1 Sample11           1      1              1                     1 Sample12           1      1              1                     1 attr(,"assign") [1] 0 1 2 3 attr(,"contrasts") attr(,"contrasts")$Age [1] "contr.treatment" attr(,"contrasts")$Genotype [1] "contr.treatment" coef=4 in design2 represents the interaction of age and genotype. Thank you for your help Joseph > sessionInfo() R version 3.0.2 Patched (2013-10-28 r64119) Platform: x86_64-apple-darwin10.8.0 (64-bit) locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats     graphics  grDevices utils     datasets  methods   base other attached packages: [1] edgeR_3.4.2  limma_3.18.7 loaded via a namespace (and not attached): [1] tools_3.0.2 [[alternative HTML version deleted]]
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@gordon-smyth
Last seen 2 hours ago
WEHI, Melbourne, Australia
Dear Joseph, You have not fitted the right model. You need ~ Genotype + Genotype:Age instead of ~ Age + Age:Genotype You can follow the section 3.3.2 in the edgeR User's Guide. Best wishes Gordon > Date: Sat, 11 Jan 2014 12:36:07 -0800 (PST) > From: joseph <jdsandjd at="" yahoo.com=""> > To: "bioconductor at r-project.org" <bioconductor at="" r-project.org=""> > Subject: [BioC] EdgeR design question? > > Hi > I am using edgeR to analyse an RNA-seq experiment with two factors, age (Young, Old) and genotype (Wt, Mutant). >> targets > ?????????? Age Genotype > Sample1? Young?????? Wt > Sample2? Young?????? Wt > Sample3? Young?????? Wt > Sample4? Young?? Mutant > Sample5? Young?? Mutant > Sample6? Young?? Mutant > Sample7??? Old?????? Wt > Sample8??? Old?????? Wt > Sample9??? Old?????? Wt > Sample10?? Old?? Mutant > Sample11?? Old?? Mutant > Sample12?? Old?? Mutant > > I know from previous experiments that the age effect is different between Wt and Mutant genotypes. > I would like to find: > 1. genes that change with age in Wt. > 2. genes that change with age in Mutant. > 3. genes that change with age differently between the genotypes (interaction). > > Can the following approach answer these questions? > > targets$Age <- relevel(targets$Age, ref="Young") > targets$Genotype <- relevel(targets$Genotype, ref="Wt") > design1 <- model.matrix(~Age + Age:Genotype, data=targets) >> design1 > ???????? (Intercept) AgeOld AgeYoung:GenotypeMutant AgeOld:GenotypeMutant > Sample1??????????? 1????? 0?????????????????????? 0???????????????????? 0 > Sample2??????????? 1????? 0?????????????????????? 0???????????????????? 0 > Sample3??????????? 1????? 0?????????????????????? 0???????????????????? 0 > Sample4??????????? 1????? 0?????????????????????? 1???????????????????? 0 > Sample5??????????? 1????? 0?????????????????????? 1???????????????????? 0 > Sample6??????????? 1????? 0?????????????????????? 1???????????????????? 0 > Sample7??????????? 1????? 1?????????????????????? 0???????????????????? 0 > Sample8??????????? 1????? 1?????????????????????? 0???????????????????? 0 > Sample9??????????? 1????? 1?????????????????????? 0???????????????????? 0 > Sample10?????????? 1????? 1?????????????????????? 0???????????????????? 1 > Sample11?????????? 1????? 1?????????????????????? 0???????????????????? 1 > Sample12?????????? 1????? 1?????????????????????? 0???????????????????? 1 > attr(,"assign") > [1] 0 1 2 2 > attr(,"contrasts") > attr(,"contrasts")$Age > [1] "contr.treatment" > attr(,"contrasts")$Genotype > [1] "contr.treatment" > > > I think coef=4 in design1 represents the genes that change with age in Mutant; but I don't know what coefficient represents the genes that change with age in Wt. > > design2 <- model.matrix(~Age*Genotype, data=targets) >> design2 > ???????? (Intercept) AgeOld GenotypeMutant AgeOld:GenotypeMutant > Sample1??????????? 1????? 0????????????? 0???????????????????? 0 > Sample2??????????? 1????? 0????????????? 0???????????????????? 0 > Sample3??????????? 1????? 0????????????? 0???????????????????? 0 > Sample4??????????? 1????? 0????????????? 1???????????????????? 0 > Sample5??????????? 1????? 0????????????? 1???????????????????? 0 > Sample6??????????? 1????? 0????????????? 1???????????????????? 0 > Sample7??????????? 1????? 1????????????? 0???????????????????? 0 > Sample8??????????? 1????? 1????????????? 0???????????????????? 0 > Sample9??????????? 1????? 1????????????? 0???????????????????? 0 > Sample10?????????? 1????? 1????????????? 1???????????????????? 1 > Sample11?????????? 1????? 1????????????? 1???????????????????? 1 > Sample12?????????? 1????? 1????????????? 1???????????????????? 1 > attr(,"assign") > [1] 0 1 2 3 > attr(,"contrasts") > attr(,"contrasts")$Age > [1] "contr.treatment" > attr(,"contrasts")$Genotype > [1] "contr.treatment" > > coef=4 in design2 represents the interaction of age and genotype. > > Thank you for your help > Joseph > > >> sessionInfo() > R version 3.0.2 Patched (2013-10-28 r64119) > Platform: x86_64-apple-darwin10.8.0 (64-bit) > > locale: > [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 > > attached base packages: > [1] stats???? graphics? grDevices utils???? datasets? methods?? base???? > > other attached packages: > [1] edgeR_3.4.2? limma_3.18.7 > > loaded via a namespace (and not attached): > [1] tools_3.0.2 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:5}}
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Dear Gordon Thank you for your help. Following the 3.3.2 section of the user guide: #set the references targets$Age <- relevel(targets$Age, ref="Young") targets$Genotype <- relevel(targets$Genotype, ref="Wt") #set design1 design1 <- model.matrix(~ Genotype + Genotype:Age, data=targets) > colnames(design1) [1] "(Intercept)"           "GenotypeMutant" "GenotypeWt:AgeOld"     "GenotypeMutant:AgeOld" #interpretation "GenotypeWt:AgeOld" (coefficient 3) represents the genes that change with age in Wt. "GenotypeMutant:AgeOld" (coefficient 4) represents the genes that change with age in Mutant. # To test for interaction, I thought I should follow section 3.3.4 of the user guide: design2 <- model.matrix(~ Genotype * Age, data=targets) > colnames(design2) [1] "(Intercept)"           "GenotypeMutant"        "AgeOld" "GenotypeMutant:AgeOld" "GenotypeMutant:AgeOld" (coefficient 4) represents represents the interaction of Genotype and Age I am doing this right? Thanks Joseph On Sunday, January 12, 2014 3:24 PM, Gordon K Smyth <smyth@wehi.edu.au> wrote: Dear Joseph, > >You have not fitted the right model.  You need > >    ~ Genotype + Genotype:Age > >instead of > >    ~ Age + Age:Genotype > >You can follow the section 3.3.2 in the edgeR User's Guide. > >Best wishes >Gordon > >> Date: Sat, 11 Jan 2014 12:36:07 -0800 (PST) >> From: joseph <jdsandjd@yahoo.com> >> To: "bioconductor@r-project.org" <bioconductor@r-project.org> >> Subject: [BioC] EdgeR design question? >> >> Hi >> I am using edgeR to analyse an RNA-seq experiment with two factors, age (Young, Old) and genotype (Wt, Mutant). >>> targets >>            Age Genotype >> Sample1  Young       Wt >> Sample2  Young       Wt >> Sample3  Young       Wt >> Sample4  Young   Mutant >> Sample5  Young   Mutant >> Sample6  Young   Mutant >> Sample7    Old       Wt >> Sample8    Old       Wt >> Sample9    Old       Wt >> Sample10   Old   Mutant >> Sample11   Old   Mutant >> Sample12   Old   Mutant >> >> I know from previous experiments that the age effect is different between Wt and Mutant genotypes. >> I would like to find: >> 1. genes that change with age in Wt. >> 2. genes that change with age in Mutant. >> 3. genes that change with age differently between the genotypes (interaction). >> >> Can the following approach answer these questions? >> >> targets$Age <- relevel(targets$Age, ref="Young") >> targets$Genotype <- relevel(targets$Genotype, ref="Wt") >> design1 <- model.matrix(~Age + Age:Genotype, data=targets) >>> design1 >>          (Intercept) AgeOld AgeYoung:GenotypeMutant AgeOld:GenotypeMutant >> Sample1            1      0                       0 0 >> Sample2            1      0                       0 0 >> Sample3            1      0                       0 0 >> Sample4            1      0                       1 0 >> Sample5            1      0                       1 0 >> Sample6            1      0                       1 0 >> Sample7            1      1                       0 0 >> Sample8            1      1                       0 0 >> Sample9            1      1                       0 0 >> Sample10           1      1                       0 1 >> Sample11           1      1                       0 1 >> Sample12           1      1                       0 1 >> attr(,"assign") >> [1] 0 1 2 2 >> attr(,"contrasts") >> attr(,"contrasts")$Age >> [1] "contr.treatment" >> attr(,"contrasts")$Genotype >> [1] "contr.treatment" >> >> >> I think coef=4 in design1 represents the genes that change with age in Mutant; but I don't know what coefficient represents the genes that change with age in Wt. >> >> design2 <- model.matrix(~Age*Genotype, data=targets) >>> design2 >>          (Intercept) AgeOld GenotypeMutant AgeOld:GenotypeMutant >> Sample1            1      0              0                     0 >> Sample2            1      0              0                     0 >> Sample3            1      0              0                     0 >> Sample4            1      0              1                     0 >> Sample5            1      0              1                     0 >> Sample6            1      0              1                     0 >> Sample7            1      1              0                     0 >> Sample8            1      1              0                     0 >> Sample9            1      1              0                     0 >> Sample10           1      1              1                     1 >> Sample11           1      1              1                     1 >> Sample12           1      1              1                     1 >> attr(,"assign") >> [1] 0 1 2 3 >> attr(,"contrasts") >> attr(,"contrasts")$Age >> [1] "contr.treatment" >> attr(,"contrasts")$Genotype >> [1] "contr.treatment" >> >> coef=4 in design2 represents the interaction of age and genotype. >> >> Thank you for your help >> Joseph >> >> >>> sessionInfo() >> R version 3.0.2 Patched (2013-10-28 r64119) >> Platform: x86_64-apple-darwin10.8.0 (64-bit) >> >> locale: >> [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 >> >> attached base packages: >> [1] stats     graphics  grDevices utils     datasets  methods base >> >> other attached packages: >> [1] edgeR_3.4.2  limma_3.18.7 >> >> loaded via a namespace (and not attached): >> [1] tools_3.0.2 > >_____________________________________________________________________ _ >The information in this email is confidential and inten...{{dropped:10}}
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Yes, that's fine. You could alternatively have tested the interaction from contrast=c(0,0,-1,1) from the first model fit. It would be the same test. Best wishes Gordon ----- Original Message ----- From: "joseph" <jdsandjd@yahoo.com> To: "Gordon K Smyth" <smyth@wehi.edu.au> Cc: "Bioconductor mailing list" <bioconductor@r-project.org> Sent: Tuesday, 14 January, 2014 4:44:55 AM Subject: Re: EdgeR design question? Dear Gordon Thank you for your help. Following the 3.3.2 section of the user guide: #set the references targets$Age <- relevel(targets$Age, ref="Young") targets$Genotype <- relevel(targets$Genotype, ref="Wt") #set design1 design1 <- model.matrix(~ Genotype + Genotype:Age, data=targets) > colnames(design1) [1] "(Intercept)" "GenotypeMutant" "GenotypeWt:AgeOld" "GenotypeMutant:AgeOld" #interpretation "GenotypeWt:AgeOld" (coefficient 3) represents the genes that change with age in Wt. "GenotypeMutant:AgeOld" (coefficient 4) represents the genes that change with age in Mutant. # To test for interaction, I thought I should follow section 3.3.4 of the user guide: design2 <- model.matrix(~ Genotype * Age, data=targets) > colnames(design2) [1] "(Intercept)" "GenotypeMutant" "AgeOld" "GenotypeMutant:AgeOld" "GenotypeMutant:AgeOld" (coefficient 4) represents represents the interaction of Genotype and Age I am doing this right? Thanks Joseph On Sunday, January 12, 2014 3:24 PM, Gordon K Smyth <smyth@wehi.edu.au> wrote: Dear Joseph, You have not fitted the right model. You need ~ Genotype + Genotype:Age instead of ~ Age + Age:Genotype You can follow the section 3.3.2 in the edgeR User's Guide. Best wishes Gordon > Date: Sat, 11 Jan 2014 12:36:07 -0800 (PST) > From: joseph < jdsandjd@yahoo.com > > To: " bioconductor@r-project.org " < bioconductor@r-project.org > > Subject: [BioC] EdgeR design question? > > Hi > I am using edgeR to analyse an RNA-seq experiment with two factors, age (Young, Old) and genotype (Wt, Mutant). >> targets > Age Genotype > Sample1 Young Wt > Sample2 Young Wt > Sample3 Young Wt > Sample4 Young Mutant > Sample5 Young Mutant > Sample6 Young Mutant > Sample7 Old Wt > Sample8 Old Wt > Sample9 Old Wt > Sample10 Old Mutant > Sample11 Old Mutant > Sample12 Old Mutant > > I know from previous experiments that the age effect is different between Wt and Mutant genotypes. > I would like to find: > 1. genes that change with age in Wt. > 2. genes that change with age in Mutant. > 3. genes that change with age differently between the genotypes (interaction). > > Can the following approach answer these questions? > > targets$Age <- relevel(targets$Age, ref="Young") > targets$Genotype <- relevel(targets$Genotype, ref="Wt") > design1 <- model.matrix(~Age + Age:Genotype, data=targets) >> design1 > (Intercept) AgeOld AgeYoung:GenotypeMutant AgeOld:GenotypeMutant > Sample1 1 0 0 0 > Sample2 1 0 0 0 > Sample3 1 0 0 0 > Sample4 1 0 1 0 > Sample5 1 0 1 0 > Sample6 1 0 1 0 > Sample7 1 1 0 0 > Sample8 1 1 0 0 > Sample9 1 1 0 0 > Sample10 1 1 0 1 > Sample11 1 1 0 1 > Sample12 1 1 0 1 > attr(,"assign") > [1] 0 1 2 2 > attr(,"contrasts") > attr(,"contrasts")$Age > [1] "contr.treatment" > attr(,"contrasts")$Genotype > [1] "contr.treatment" > > > I think coef=4 in design1 represents the genes that change with age in Mutant; but I don't know what coefficient represents the genes that change with age in Wt. > > design2 <- model.matrix(~Age*Genotype, data=targets) >> design2 > (Intercept) AgeOld GenotypeMutant AgeOld:GenotypeMutant > Sample1 1 0 0 0 > Sample2 1 0 0 0 > Sample3 1 0 0 0 > Sample4 1 0 1 0 > Sample5 1 0 1 0 > Sample6 1 0 1 0 > Sample7 1 1 0 0 > Sample8 1 1 0 0 > Sample9 1 1 0 0 > Sample10 1 1 1 1 > Sample11 1 1 1 1 > Sample12 1 1 1 1 > attr(,"assign") > [1] 0 1 2 3 > attr(,"contrasts") > attr(,"contrasts")$Age > [1] "contr.treatment" > attr(,"contrasts")$Genotype > [1] "contr.treatment" > > coef=4 in design2 represents the interaction of age and genotype. > > Thank you for your help > Joseph > > >> sessionInfo() > R version 3.0.2 Patched (2013-10-28 r64119) > Platform: x86_64-apple-darwin10.8.0 (64-bit) > > locale: > [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] edgeR_3.4.2 limma_3.18.7 > > loaded via a namespace (and not attached): > [1] tools_3.0.2 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:17}}
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