Analysis affymetrix of experiment....help please
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suparna mitra ▴ 290
@suparna-mitra-5328
Last seen 9.7 years ago
Hello Group, I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using BiC. Previously i was using different softwares for this. And this is my first try with Bioconductor for big experiment. So thought to get some advice in the beginning. I have Three groups of patient: (In-vivo) A-Acute reaction. Patient taking a drug X develops reaction. R-recovered (6 weeks after acute reaction-not longer taking drug X). T-Tolerant. Patient on X and tolerating treatment. Now in in-vitro study we used another constant Y RXY recovered and challenged with X+Y RY recovered challenged with only Y. RXY vs RY are to exclude effects by Y. TXY tolerant and challenged with X+Y, TY tolerant challenged with only Y. TXY vs TY are to exclude effects by Y. No I want to check the cross relation and effects A vs R, RvsT and Avs T and differentially expressed genes for each comparison. And the same in invitro. There are not same patients in different groups, thus I think I want to apply unpaired-t test. This is what I tried: > sessionInfo() R version 2.15.1 (2012-06-22) Platform: i386-apple-darwin9.8.0/i386 (32-bit) locale: [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] statmod_1.4.15 limma_3.12.1 annotate_1.34.1 hugene10stprobeset.db_8.0.1 org.Hs.eg.db_2.7.1 [6] BiocInstaller_1.4.7 affycoretools_1.28.0 KEGG.db_2.7.1 GO.db_2.7.1 AnnotationDbi_1.18.1 [11] affy_1.34.0 Biobase_2.16.0 BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 [16] DBI_0.2-5 oligo_1.20.4 oligoClasses_1.18.0 rmaOligoinvivo = oligo::rma(InVivodat1) Background correcting Normalizing Calculating Expression > rmaOligoinvitro = oligo::rma(InVitrodat1) Background correcting Normalizing Calculating Expression > maplot(rmaOligoinvivo) > maplot(rmaOligoinvitro) > InVivoTargets FileName Treatment 1 MC1 A 2 MC2 A 3 MC3 A 4 MC4 A 5 MC5 A 6 MC6 A 7 MC7 R 8 MC8 R 9 MC9 R 10 MC10 R 11 MC11 R 12 MC12 R 13 MC13 T 14 MC14 T 15 MC15 T 16 MC16 T 17 MC17 T 18 MC18 T > InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") > InVitroTargets FileName Treatment Batch CD4 1 MC19 RY 1 High 2 MC20 TY 1 Low 3 MC21 RY 2 High 4 MC22 TY 2 High 5 MC23 TY 2 Low 6 MC24 RY 2 High 7 MC25 TXY 1 Low 8 MC26 RXY 1 High 9 MC27 RXY 2 Low 10 MC28 TXY 2 High 11 MC29 RXY 2 High 12 MC30 TXY 2 High f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) design.invivo <- model.matrix(~0 + f.invivo) > > colnames(design.invivo) <- c("A", "R", "T") > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = design.invivo) > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > fit2.invivo <-eBayes(fit2.invivo) > topTable(fit2.invivo, coef = 1, adjust = "fdr") ID logFC AveExpr t P.Value adj.P.Val B 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 -2.323922 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 -2.454618 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 -2.607991 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 -2.685960 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 -2.711203 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 -2.800385 I am progressing in a right way? Further I want to perform unpaired t test for comparing AvsT and so on. Any help will be really great. Thanks a lot , Mitra. -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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@sean-davis-490
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On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > Hello Group, > I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using BiC. > Previously i was using different softwares for this. And this is my first > try with Bioconductor for big experiment. So thought to get some advice in > the beginning. > I have Three groups of patient: (In-vivo) > A-Acute reaction. Patient taking a drug X develops reaction. > R-recovered (6 weeks after acute reaction-not longer taking drug X). > T-Tolerant. Patient on X and tolerating treatment. > > Now in in-vitro study we used another constant Y > RXY recovered and challenged with X+Y > RY recovered challenged with only Y. RXY vs RY are to exclude effects by > Y. > TXY tolerant and challenged with X+Y, > TY tolerant challenged with only Y. TXY vs TY are to exclude effects by > Y. > > No I want to check the cross relation and effects A vs R, RvsT and Avs T > and differentially expressed genes for each comparison. And the same in > invitro. There are not same patients in different groups, thus I think I > want to apply unpaired-t test. > > This is what I tried: > > sessionInfo() > R version 2.15.1 (2012-06-22) > Platform: i386-apple-darwin9.8.0/i386 (32-bit) > > locale: > [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] statmod_1.4.15 limma_3.12.1 > annotate_1.34.1 hugene10stprobeset.db_8.0.1 org.Hs.eg.db_2.7.1 > > [6] BiocInstaller_1.4.7 affycoretools_1.28.0 KEGG.db_2.7.1 > GO.db_2.7.1 AnnotationDbi_1.18.1 > [11] affy_1.34.0 Biobase_2.16.0 > BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 > > [16] DBI_0.2-5 oligo_1.20.4 > oligoClasses_1.18.0 > > > rmaOligoinvivo = oligo::rma(InVivodat1) > Background correcting > Normalizing > Calculating Expression > > > rmaOligoinvitro = oligo::rma(InVitrodat1) > Background correcting > Normalizing > Calculating Expression > > > maplot(rmaOligoinvivo) > > maplot(rmaOligoinvitro) > > InVivoTargets > FileName Treatment > 1 MC1 A > 2 MC2 A > 3 MC3 A > 4 MC4 A > 5 MC5 A > 6 MC6 A > 7 MC7 R > 8 MC8 R > 9 MC9 R > 10 MC10 R > 11 MC11 R > 12 MC12 R > 13 MC13 T > 14 MC14 T > 15 MC15 T > 16 MC16 T > 17 MC17 T > 18 MC18 T > > > > InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") > > InVitroTargets > FileName Treatment Batch CD4 > 1 MC19 RY 1 High > 2 MC20 TY 1 Low > 3 MC21 RY 2 High > 4 MC22 TY 2 High > 5 MC23 TY 2 Low > 6 MC24 RY 2 High > 7 MC25 TXY 1 Low > 8 MC26 RXY 1 High > 9 MC27 RXY 2 Low > 10 MC28 TXY 2 High > 11 MC29 RXY 2 High > 12 MC30 TXY 2 High > > f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) > > design.invivo <- model.matrix(~0 + f.invivo) > > > > > > colnames(design.invivo) <- c("A", "R", "T") > > > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > > > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = > design.invivo) > > > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > > > fit2.invivo <-eBayes(fit2.invivo) > > > topTable(fit2.invivo, coef = 1, adjust = "fdr") > > ID logFC AveExpr t P.Value adj.P.Val > B > > 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > -2.323922 > > 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > -2.454618 > > 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > -2.607991 > > 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > -2.685960 > > 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > -2.711203 > > 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > -2.800385 > I am progressing in a right way? Further I want to perform unpaired t test > for comparing AvsT and so on. Any help will be really great. > Hi, Mitra. I think that looks about right. You have already performed the unpaired t-test of AvsT (well, actually TvsA, but the p-values will be the same) as coefficient 3. Sean [[alternative HTML version deleted]]
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Dear Sean, I have been reading Bioconductor and limma user guide and thus this is I tried. But just being a novice, wanted to make sure if I am right. I know I have perform t-test when I created the contrast, but can you please help me how can I perform unpaired t-test here. I am concerned as the patients in groups are not same. Thanks, Mitra On 7 September 2012 11:41, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > >> Hello Group, >> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using BiC. >> Previously i was using different softwares for this. And this is my first >> try with Bioconductor for big experiment. So thought to get some advice in >> the beginning. >> I have Three groups of patient: (In-vivo) >> A-Acute reaction. Patient taking a drug X develops reaction. >> R-recovered (6 weeks after acute reaction-not longer taking drug X). >> T-Tolerant. Patient on X and tolerating treatment. >> >> Now in in-vitro study we used another constant Y >> RXY recovered and challenged with X+Y >> RY recovered challenged with only Y. RXY vs RY are to exclude effects >> by >> Y. >> TXY tolerant and challenged with X+Y, >> TY tolerant challenged with only Y. TXY vs TY are to exclude effects by >> Y. >> >> No I want to check the cross relation and effects A vs R, RvsT and Avs T >> and differentially expressed genes for each comparison. And the same in >> invitro. There are not same patients in different groups, thus I think I >> want to apply unpaired-t test. >> >> This is what I tried: >> > sessionInfo() >> R version 2.15.1 (2012-06-22) >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >> >> locale: >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 >> >> attached base packages: >> [1] stats graphics grDevices utils datasets methods base >> >> other attached packages: >> [1] statmod_1.4.15 limma_3.12.1 >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >> org.Hs.eg.db_2.7.1 >> >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 KEGG.db_2.7.1 >> GO.db_2.7.1 AnnotationDbi_1.18.1 >> [11] affy_1.34.0 Biobase_2.16.0 >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 >> >> [16] DBI_0.2-5 oligo_1.20.4 >> oligoClasses_1.18.0 >> >> >> rmaOligoinvivo = oligo::rma(InVivodat1) >> Background correcting >> Normalizing >> Calculating Expression >> >> > rmaOligoinvitro = oligo::rma(InVitrodat1) >> Background correcting >> Normalizing >> Calculating Expression >> >> > maplot(rmaOligoinvivo) >> > maplot(rmaOligoinvitro) >> > InVivoTargets >> FileName Treatment >> 1 MC1 A >> 2 MC2 A >> 3 MC3 A >> 4 MC4 A >> 5 MC5 A >> 6 MC6 A >> 7 MC7 R >> 8 MC8 R >> 9 MC9 R >> 10 MC10 R >> 11 MC11 R >> 12 MC12 R >> 13 MC13 T >> 14 MC14 T >> 15 MC15 T >> 16 MC16 T >> 17 MC17 T >> 18 MC18 T >> > >> >> InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") >> > InVitroTargets >> FileName Treatment Batch CD4 >> 1 MC19 RY 1 High >> 2 MC20 TY 1 Low >> 3 MC21 RY 2 High >> 4 MC22 TY 2 High >> 5 MC23 TY 2 Low >> 6 MC24 RY 2 High >> 7 MC25 TXY 1 Low >> 8 MC26 RXY 1 High >> 9 MC27 RXY 2 Low >> 10 MC28 TXY 2 High >> 11 MC29 RXY 2 High >> 12 MC30 TXY 2 High >> >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) >> >> design.invivo <- model.matrix(~0 + f.invivo) >> >> > >> >> > colnames(design.invivo) <- c("A", "R", "T") >> >> > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) >> >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = >> design.invivo) >> >> > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) >> >> > fit2.invivo <-eBayes(fit2.invivo) >> >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") >> >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >> -2.323922 >> >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >> -2.454618 >> >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >> -2.607991 >> >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >> -2.685960 >> >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >> -2.711203 >> >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >> -2.800385 >> I am progressing in a right way? Further I want to perform unpaired t test >> for comparing AvsT and so on. Any help will be really great. >> > > Hi, Mitra. I think that looks about right. You have already performed > the unpaired t-test of AvsT (well, actually TvsA, but the p-values will be > the same) as coefficient 3. > > Sean > > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > Dear Sean, > I have been reading Bioconductor and limma user guide and thus this is I > tried. > But just being a novice, wanted to make sure if I am right. > I know I have perform t-test when I created the contrast, but can you > please help me how can I perform unpaired t-test here. I am concerned as > the patients in groups are not same. > The t-test you performed was unpaired; unpaired is the "default". Sean > Thanks, > Mitra > > On 7 September 2012 11:41, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > > > > > On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra <smitra@liverpool.ac.uk> >wrote: > > > >> Hello Group, > >> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using BiC. > >> Previously i was using different softwares for this. And this is my > first > >> try with Bioconductor for big experiment. So thought to get some advice > in > >> the beginning. > >> I have Three groups of patient: (In-vivo) > >> A-Acute reaction. Patient taking a drug X develops reaction. > >> R-recovered (6 weeks after acute reaction-not longer taking drug X). > >> T-Tolerant. Patient on X and tolerating treatment. > >> > >> Now in in-vitro study we used another constant Y > >> RXY recovered and challenged with X+Y > >> RY recovered challenged with only Y. RXY vs RY are to exclude effects > >> by > >> Y. > >> TXY tolerant and challenged with X+Y, > >> TY tolerant challenged with only Y. TXY vs TY are to exclude effects > by > >> Y. > >> > >> No I want to check the cross relation and effects A vs R, RvsT and Avs T > >> and differentially expressed genes for each comparison. And the same in > >> invitro. There are not same patients in different groups, thus I think I > >> want to apply unpaired-t test. > >> > >> This is what I tried: > >> > sessionInfo() > >> R version 2.15.1 (2012-06-22) > >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) > >> > >> locale: > >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 > >> > >> attached base packages: > >> [1] stats graphics grDevices utils datasets methods base > >> > >> other attached packages: > >> [1] statmod_1.4.15 limma_3.12.1 > >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 > >> org.Hs.eg.db_2.7.1 > >> > >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 > KEGG.db_2.7.1 > >> GO.db_2.7.1 AnnotationDbi_1.18.1 > >> [11] affy_1.34.0 Biobase_2.16.0 > >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 > >> > >> [16] DBI_0.2-5 oligo_1.20.4 > >> oligoClasses_1.18.0 > >> > >> > >> rmaOligoinvivo = oligo::rma(InVivodat1) > >> Background correcting > >> Normalizing > >> Calculating Expression > >> > >> > rmaOligoinvitro = oligo::rma(InVitrodat1) > >> Background correcting > >> Normalizing > >> Calculating Expression > >> > >> > maplot(rmaOligoinvivo) > >> > maplot(rmaOligoinvitro) > >> > InVivoTargets > >> FileName Treatment > >> 1 MC1 A > >> 2 MC2 A > >> 3 MC3 A > >> 4 MC4 A > >> 5 MC5 A > >> 6 MC6 A > >> 7 MC7 R > >> 8 MC8 R > >> 9 MC9 R > >> 10 MC10 R > >> 11 MC11 R > >> 12 MC12 R > >> 13 MC13 T > >> 14 MC14 T > >> 15 MC15 T > >> 16 MC16 T > >> 17 MC17 T > >> 18 MC18 T > >> > > >> > >> > InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") > >> > InVitroTargets > >> FileName Treatment Batch CD4 > >> 1 MC19 RY 1 High > >> 2 MC20 TY 1 Low > >> 3 MC21 RY 2 High > >> 4 MC22 TY 2 High > >> 5 MC23 TY 2 Low > >> 6 MC24 RY 2 High > >> 7 MC25 TXY 1 Low > >> 8 MC26 RXY 1 High > >> 9 MC27 RXY 2 Low > >> 10 MC28 TXY 2 High > >> 11 MC29 RXY 2 High > >> 12 MC30 TXY 2 High > >> > >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) > >> > >> design.invivo <- model.matrix(~0 + f.invivo) > >> > >> > > >> > >> > colnames(design.invivo) <- c("A", "R", "T") > >> > >> > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > >> > >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = > >> design.invivo) > >> > >> > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > >> > >> > fit2.invivo <-eBayes(fit2.invivo) > >> > >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") > >> > >> ID logFC AveExpr t P.Value adj.P.Val > >> B > >> > >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > >> -2.323922 > >> > >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > >> -2.454618 > >> > >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > >> -2.607991 > >> > >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > >> -2.685960 > >> > >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > >> -2.711203 > >> > >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > >> -2.800385 > >> I am progressing in a right way? Further I want to perform unpaired t > test > >> for comparing AvsT and so on. Any help will be really great. > >> > > > > Hi, Mitra. I think that looks about right. You have already performed > > the unpaired t-test of AvsT (well, actually TvsA, but the p-values will > be > > the same) as coefficient 3. > > > > Sean > > > > > > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor@r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]
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Oh thanks.. I missed this point. But can you suggest me one more thing... when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the same result as adjust = "fdr". for topTable. Is it normal? Further when I tried to do vennDiagram I was surprized to see 0 in all circles. Thus I thought I must be doing something wrong. Sorry if my question is silly. Here is what I tried. > topTable(fit2.invivo, coef = 1, adjust = "fdr") ID logFC AveExpr t P.Value adj.P.Val B 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 -2.323922 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 -2.454618 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 -2.607991 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 -2.685960 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 -2.711203 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 -2.800385 > topTable(fit2.invivo, coef = 2, adjust = "fdr") ID logFC AveExpr t P.Value adj.P.Val B 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 -2.469821 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 -2.652426 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 -2.662042 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 -2.702782 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 -2.707492 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 -2.717698 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 -2.734873 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 -2.772143 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 -2.779941 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 -2.785214 > topTable(fit2.invivo, coef = 3, adjust = "fdr") ID logFC AveExpr t P.Value adj.P.Val B 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 -0.6982314 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 -1.2472882 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 -1.2727646 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 -1.2831553 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 -1.4031177 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 -1.4733549 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 -1.5025187 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 -1.5612836 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 -1.6535466 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 -1.6662493 > > > results <- decideTests(fit2.invivo) > vennDiagram(results) see the plot attached. Thanks, Mitra On 7 September 2012 12:03, Sean Davis <sdavis2 at="" mail.nih.gov=""> wrote: > On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra <smitra at="" liverpool.ac.uk=""> >wrote: > > > Dear Sean, > > I have been reading Bioconductor and limma user guide and thus this is > I > > tried. > > But just being a novice, wanted to make sure if I am right. > > I know I have perform t-test when I created the contrast, but can you > > please help me how can I perform unpaired t-test here. I am concerned as > > the patients in groups are not same. > > > > > The t-test you performed was unpaired; unpaired is the "default". > > Sean > > > > Thanks, > > Mitra > > > > On 7 September 2012 11:41, Sean Davis <sdavis2 at="" mail.nih.gov=""> wrote: > > > > > > > > > > > On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra <smitra at="" liverpool.ac.uk=""> > >wrote: > > > > > >> Hello Group, > > >> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using BiC. > > >> Previously i was using different softwares for this. And this is my > > first > > >> try with Bioconductor for big experiment. So thought to get some > advice > > in > > >> the beginning. > > >> I have Three groups of patient: (In-vivo) > > >> A-Acute reaction. Patient taking a drug X develops reaction. > > >> R-recovered (6 weeks after acute reaction-not longer taking drug X). > > >> T-Tolerant. Patient on X and tolerating treatment. > > >> > > >> Now in in-vitro study we used another constant Y > > >> RXY recovered and challenged with X+Y > > >> RY recovered challenged with only Y. RXY vs RY are to exclude > effects > > >> by > > >> Y. > > >> TXY tolerant and challenged with X+Y, > > >> TY tolerant challenged with only Y. TXY vs TY are to exclude effects > > by > > >> Y. > > >> > > >> No I want to check the cross relation and effects A vs R, RvsT and > Avs T > > >> and differentially expressed genes for each comparison. And the same > in > > >> invitro. There are not same patients in different groups, thus I > think I > > >> want to apply unpaired-t test. > > >> > > >> This is what I tried: > > >> > sessionInfo() > > >> R version 2.15.1 (2012-06-22) > > >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) > > >> > > >> locale: > > >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 > > >> > > >> attached base packages: > > >> [1] stats graphics grDevices utils datasets methods base > > >> > > >> other attached packages: > > >> [1] statmod_1.4.15 limma_3.12.1 > > >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 > > >> org.Hs.eg.db_2.7.1 > > >> > > >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 > > KEGG.db_2.7.1 > > >> GO.db_2.7.1 AnnotationDbi_1.18.1 > > >> [11] affy_1.34.0 Biobase_2.16.0 > > >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 > RSQLite_0.11.1 > > >> > > >> [16] DBI_0.2-5 oligo_1.20.4 > > >> oligoClasses_1.18.0 > > >> > > >> > > >> rmaOligoinvivo = oligo::rma(InVivodat1) > > >> Background correcting > > >> Normalizing > > >> Calculating Expression > > >> > > >> > rmaOligoinvitro = oligo::rma(InVitrodat1) > > >> Background correcting > > >> Normalizing > > >> Calculating Expression > > >> > > >> > maplot(rmaOligoinvivo) > > >> > maplot(rmaOligoinvitro) > > >> > InVivoTargets > > >> FileName Treatment > > >> 1 MC1 A > > >> 2 MC2 A > > >> 3 MC3 A > > >> 4 MC4 A > > >> 5 MC5 A > > >> 6 MC6 A > > >> 7 MC7 R > > >> 8 MC8 R > > >> 9 MC9 R > > >> 10 MC10 R > > >> 11 MC11 R > > >> 12 MC12 R > > >> 13 MC13 T > > >> 14 MC14 T > > >> 15 MC15 T > > >> 16 MC16 T > > >> 17 MC17 T > > >> 18 MC18 T > > >> > > > >> > > >> > > > InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") > > >> > InVitroTargets > > >> FileName Treatment Batch CD4 > > >> 1 MC19 RY 1 High > > >> 2 MC20 TY 1 Low > > >> 3 MC21 RY 2 High > > >> 4 MC22 TY 2 High > > >> 5 MC23 TY 2 Low > > >> 6 MC24 RY 2 High > > >> 7 MC25 TXY 1 Low > > >> 8 MC26 RXY 1 High > > >> 9 MC27 RXY 2 Low > > >> 10 MC28 TXY 2 High > > >> 11 MC29 RXY 2 High > > >> 12 MC30 TXY 2 High > > >> > > >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) > > >> > > >> design.invivo <- model.matrix(~0 + f.invivo) > > >> > > >> > > > >> > > >> > colnames(design.invivo) <- c("A", "R", "T") > > >> > > >> > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > > >> > > >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = > > >> design.invivo) > > >> > > >> > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > > >> > > >> > fit2.invivo <-eBayes(fit2.invivo) > > >> > > >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") > > >> > > >> ID logFC AveExpr t P.Value adj.P.Val > > >> B > > >> > > >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > >> -2.023533 > > >> > > >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > >> -2.023533 > > >> > > >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > >> -2.023533 > > >> > > >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > >> -2.023533 > > >> > > >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > > >> -2.323922 > > >> > > >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > > >> -2.454618 > > >> > > >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > > >> -2.607991 > > >> > > >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > > >> -2.685960 > > >> > > >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > > >> -2.711203 > > >> > > >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > > >> -2.800385 > > >> I am progressing in a right way? Further I want to perform unpaired t > > test > > >> for comparing AvsT and so on. Any help will be really great. > > >> > > > > > > Hi, Mitra. I think that looks about right. You have already performed > > > the unpaired t-test of AvsT (well, actually TvsA, but the p-values will > > be > > > the same) as coefficient 3. > > > > > > Sean > > > > > > > > > > > > > > -- > > Dr. Suparna Mitra > > Wolfson Centre for Personalised Medicine > > Department of Molecular and Clinical Pharmacology > > Institute of Translational Medicine University of Liverpool > > Block A: Waterhouse Buildings, L69 3GL Liverpool > > > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > > M: +44 (0) 7511387895 > > Email id: smitra at liverpool.ac.uk > > Alternative Email id: suparna.mitra.sm at gmail.com > > > > [[alternative HTML version deleted]] > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor at r-project.org > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: > > http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra at liverpool.ac.uk Alternative Email id: suparna.mitra.sm at gmail.com -------------- next part -------------- A non-text attachment was scrubbed... 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Entering edit mode
On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > Oh thanks.. I missed this point. But can you suggest me one more thing... > when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the same > result as adjust = "fdr". for topTable. Is it normal? > Yes. They are the same. See the help for p.adjust for details. > Further when I tried to do vennDiagram I was surprized to see 0 in all > circles. Thus I thought I must be doing something wrong. Sorry if my > question is silly. > Unfortunately, you have no significantly differentially-expressed genes. Note that all of the adjusted p-values are pretty high. You can try to filter your genes based on variance before testing to try to reduce the number of genes entering your test and multiple correction. However, having worked with this kind of biological system (patients), you may suffering from a problem of a small biological effect in the setting of large biological variation. A larger sample size may be necessary. Sean > Here is what I tried. > > > topTable(fit2.invivo, coef = 1, adjust = "fdr") > > ID logFC AveExpr t P.Value adj.P.Val > B > > 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > -2.323922 > > 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > -2.454618 > > 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > -2.607991 > > 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > -2.685960 > > 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > -2.711203 > > 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > -2.800385 > > > topTable(fit2.invivo, coef = 2, adjust = "fdr") > > ID logFC AveExpr t P.Value adj.P.Val > B > > 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 > -2.469821 > > 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 > -2.652426 > > 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 > -2.662042 > > 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 > -2.702782 > > 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 > -2.707492 > > 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 > -2.717698 > > 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 > -2.734873 > > 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 > -2.772143 > > 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 > -2.779941 > > 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 > -2.785214 > > > topTable(fit2.invivo, coef = 3, adjust = "fdr") > > ID logFC AveExpr t P.Value adj.P.Val > B > > 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 > -0.6982314 > > 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 > -1.2472882 > > 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 > -1.2727646 > > 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 > -1.2831553 > > 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 > -1.4031177 > > 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 > -1.4733549 > > 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 > -1.5025187 > > 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 > -1.5612836 > > 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 > -1.6535466 > > 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 > -1.6662493 > > > > > > > > > results <- decideTests(fit2.invivo) > > > vennDiagram(results) > see the plot attached. > Thanks, > Mitra > > > On 7 September 2012 12:03, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra <smitra@liverpool.ac.uk> > >wrote: > > > > > Dear Sean, > > > I have been reading Bioconductor and limma user guide and thus this > is > > I > > > tried. > > > But just being a novice, wanted to make sure if I am right. > > > I know I have perform t-test when I created the contrast, but can you > > > please help me how can I perform unpaired t-test here. I am concerned > as > > > the patients in groups are not same. > > > > > > > > > The t-test you performed was unpaired; unpaired is the "default". > > > > Sean > > > > > > > Thanks, > > > Mitra > > > > > > On 7 September 2012 11:41, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > > > > > > > > > > > > > On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra < > smitra@liverpool.ac.uk > > > >wrote: > > > > > > > >> Hello Group, > > > >> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using > BiC. > > > >> Previously i was using different softwares for this. And this is my > > > first > > > >> try with Bioconductor for big experiment. So thought to get some > > advice > > > in > > > >> the beginning. > > > >> I have Three groups of patient: (In-vivo) > > > >> A-Acute reaction. Patient taking a drug X develops reaction. > > > >> R-recovered (6 weeks after acute reaction-not longer taking drug > X). > > > >> T-Tolerant. Patient on X and tolerating treatment. > > > >> > > > >> Now in in-vitro study we used another constant Y > > > >> RXY recovered and challenged with X+Y > > > >> RY recovered challenged with only Y. RXY vs RY are to exclude > > effects > > > >> by > > > >> Y. > > > >> TXY tolerant and challenged with X+Y, > > > >> TY tolerant challenged with only Y. TXY vs TY are to exclude > effects > > > by > > > >> Y. > > > >> > > > >> No I want to check the cross relation and effects A vs R, RvsT and > > Avs T > > > >> and differentially expressed genes for each comparison. And the > same > > in > > > >> invitro. There are not same patients in different groups, thus I > > think I > > > >> want to apply unpaired-t test. > > > >> > > > >> This is what I tried: > > > >> > sessionInfo() > > > >> R version 2.15.1 (2012-06-22) > > > >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) > > > >> > > > >> locale: > > > >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 > > > >> > > > >> attached base packages: > > > >> [1] stats graphics grDevices utils datasets methods base > > > >> > > > >> other attached packages: > > > >> [1] statmod_1.4.15 limma_3.12.1 > > > >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 > > > >> org.Hs.eg.db_2.7.1 > > > >> > > > >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 > > > KEGG.db_2.7.1 > > > >> GO.db_2.7.1 AnnotationDbi_1.18.1 > > > >> [11] affy_1.34.0 Biobase_2.16.0 > > > >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 > > RSQLite_0.11.1 > > > >> > > > >> [16] DBI_0.2-5 oligo_1.20.4 > > > >> oligoClasses_1.18.0 > > > >> > > > >> > > > >> rmaOligoinvivo = oligo::rma(InVivodat1) > > > >> Background correcting > > > >> Normalizing > > > >> Calculating Expression > > > >> > > > >> > rmaOligoinvitro = oligo::rma(InVitrodat1) > > > >> Background correcting > > > >> Normalizing > > > >> Calculating Expression > > > >> > > > >> > maplot(rmaOligoinvivo) > > > >> > maplot(rmaOligoinvitro) > > > >> > InVivoTargets > > > >> FileName Treatment > > > >> 1 MC1 A > > > >> 2 MC2 A > > > >> 3 MC3 A > > > >> 4 MC4 A > > > >> 5 MC5 A > > > >> 6 MC6 A > > > >> 7 MC7 R > > > >> 8 MC8 R > > > >> 9 MC9 R > > > >> 10 MC10 R > > > >> 11 MC11 R > > > >> 12 MC12 R > > > >> 13 MC13 T > > > >> 14 MC14 T > > > >> 15 MC15 T > > > >> 16 MC16 T > > > >> 17 MC17 T > > > >> 18 MC18 T > > > >> > > > > >> > > > >> > > > > > > InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") > > > >> > InVitroTargets > > > >> FileName Treatment Batch CD4 > > > >> 1 MC19 RY 1 High > > > >> 2 MC20 TY 1 Low > > > >> 3 MC21 RY 2 High > > > >> 4 MC22 TY 2 High > > > >> 5 MC23 TY 2 Low > > > >> 6 MC24 RY 2 High > > > >> 7 MC25 TXY 1 Low > > > >> 8 MC26 RXY 1 High > > > >> 9 MC27 RXY 2 Low > > > >> 10 MC28 TXY 2 High > > > >> 11 MC29 RXY 2 High > > > >> 12 MC30 TXY 2 High > > > >> > > > >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", > "T")) > > > >> > > > >> design.invivo <- model.matrix(~0 + f.invivo) > > > >> > > > >> > > > > >> > > > >> > colnames(design.invivo) <- c("A", "R", "T") > > > >> > > > >> > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > > > >> > > > >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = > > > >> design.invivo) > > > >> > > > >> > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > > > >> > > > >> > fit2.invivo <-eBayes(fit2.invivo) > > > >> > > > >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") > > > >> > > > >> ID logFC AveExpr t P.Value adj.P.Val > > > >> B > > > >> > > > >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > > >> -2.023533 > > > >> > > > >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > > >> -2.023533 > > > >> > > > >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > > >> -2.023533 > > > >> > > > >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > > > >> -2.023533 > > > >> > > > >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > > > >> -2.323922 > > > >> > > > >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > > > >> -2.454618 > > > >> > > > >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > > > >> -2.607991 > > > >> > > > >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > > > >> -2.685960 > > > >> > > > >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > > > >> -2.711203 > > > >> > > > >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > > > >> -2.800385 > > > >> I am progressing in a right way? Further I want to perform unpaired > t > > > test > > > >> for comparing AvsT and so on. Any help will be really great. > > > >> > > > > > > > > Hi, Mitra. I think that looks about right. You have already > performed > > > > the unpaired t-test of AvsT (well, actually TvsA, but the p-values > will > > > be > > > > the same) as coefficient 3. > > > > > > > > Sean > > > > > > > > > > > > > > > > > > > > -- > > > Dr. Suparna Mitra > > > Wolfson Centre for Personalised Medicine > > > Department of Molecular and Clinical Pharmacology > > > Institute of Translational Medicine University of Liverpool > > > Block A: Waterhouse Buildings, L69 3GL Liverpool > > > > > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > > > M: +44 (0) 7511387895 > > > Email id: smitra@liverpool.ac.uk > > > Alternative Email id: suparna.mitra.sm@gmail.com > > > > > > [[alternative HTML version deleted]] > > > > > > _______________________________________________ > > > Bioconductor mailing list > > > Bioconductor@r-project.org > > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > > Search the archives: > > > http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > > > > [[alternative HTML version deleted]] > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@r-project.org > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: > > http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > _______________________________________________ > Bioconductor mailing list > Bioconductor@r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]
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On 9/7/2012 7:26 AM, Sean Davis wrote: > On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra<smitra at="" liverpool.ac.uk="">wrote: > >> Oh thanks.. I missed this point. But can you suggest me one more thing... >> when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the same >> result as adjust = "fdr". for topTable. Is it normal? >> > Yes. They are the same. See the help for p.adjust for details. > > >> Further when I tried to do vennDiagram I was surprized to see 0 in all >> circles. Thus I thought I must be doing something wrong. Sorry if my >> question is silly. >> > Unfortunately, you have no significantly differentially-expressed genes. > Note that all of the adjusted p-values are pretty high. You can try to > filter your genes based on variance before testing to try to reduce the > number of genes entering your test and multiple correction. However, > having worked with this kind of biological system (patients), you may > suffering from a problem of a small biological effect in the setting of > large biological variation. A larger sample size may be necessary. You may also be suffering from large technical variation, which could be helped by applying array weights. See ?arrayWeights for more information. Best, Jim > > Sean > > >> Here is what I tried. >> >>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >> -2.323922 >> >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >> -2.454618 >> >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >> -2.607991 >> >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >> -2.685960 >> >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >> -2.711203 >> >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >> -2.800385 >> >>> topTable(fit2.invivo, coef = 2, adjust = "fdr") >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 >> -2.469821 >> >> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 >> -2.652426 >> >> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 >> -2.662042 >> >> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 >> -2.702782 >> >> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 >> -2.707492 >> >> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 >> -2.717698 >> >> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 >> -2.734873 >> >> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 >> -2.772143 >> >> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 >> -2.779941 >> >> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 >> -2.785214 >> >>> topTable(fit2.invivo, coef = 3, adjust = "fdr") >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 >> -0.6982314 >> >> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 >> -1.2472882 >> >> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 >> -1.2727646 >> >> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 >> -1.2831553 >> >> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 >> -1.4031177 >> >> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 >> -1.4733549 >> >> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 >> -1.5025187 >> >> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 >> -1.5612836 >> >> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 >> -1.6535466 >> >> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 >> -1.6662493 >> >>> results<- decideTests(fit2.invivo) >>> vennDiagram(results) >> see the plot attached. >> Thanks, >> Mitra >> >> >> On 7 September 2012 12:03, Sean Davis<sdavis2 at="" mail.nih.gov=""> wrote: >> >>> On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra<smitra at="" liverpool.ac.uk="">>>> wrote: >>>> Dear Sean, >>>> I have been reading Bioconductor and limma user guide and thus this >> is >>> I >>>> tried. >>>> But just being a novice, wanted to make sure if I am right. >>>> I know I have perform t-test when I created the contrast, but can you >>>> please help me how can I perform unpaired t-test here. I am concerned >> as >>>> the patients in groups are not same. >>>> >>> >>> The t-test you performed was unpaired; unpaired is the "default". >>> >>> Sean >>> >>> >>>> Thanks, >>>> Mitra >>>> >>>> On 7 September 2012 11:41, Sean Davis<sdavis2 at="" mail.nih.gov=""> wrote: >>>> >>>>> >>>>> On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra< >> smitra at liverpool.ac.uk >>>>> wrote: >>>>> >>>>>> Hello Group, >>>>>> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using >> BiC. >>>>>> Previously i was using different softwares for this. And this is my >>>> first >>>>>> try with Bioconductor for big experiment. So thought to get some >>> advice >>>> in >>>>>> the beginning. >>>>>> I have Three groups of patient: (In-vivo) >>>>>> A-Acute reaction. Patient taking a drug X develops reaction. >>>>>> R-recovered (6 weeks after acute reaction-not longer taking drug >> X). >>>>>> T-Tolerant. Patient on X and tolerating treatment. >>>>>> >>>>>> Now in in-vitro study we used another constant Y >>>>>> RXY recovered and challenged with X+Y >>>>>> RY recovered challenged with only Y. RXY vs RY are to exclude >>> effects >>>>>> by >>>>>> Y. >>>>>> TXY tolerant and challenged with X+Y, >>>>>> TY tolerant challenged with only Y. TXY vs TY are to exclude >> effects >>>> by >>>>>> Y. >>>>>> >>>>>> No I want to check the cross relation and effects A vs R, RvsT and >>> Avs T >>>>>> and differentially expressed genes for each comparison. And the >> same >>> in >>>>>> invitro. There are not same patients in different groups, thus I >>> think I >>>>>> want to apply unpaired-t test. >>>>>> >>>>>> This is what I tried: >>>>>>> sessionInfo() >>>>>> R version 2.15.1 (2012-06-22) >>>>>> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >>>>>> >>>>>> locale: >>>>>> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 >>>>>> >>>>>> attached base packages: >>>>>> [1] stats graphics grDevices utils datasets methods base >>>>>> >>>>>> other attached packages: >>>>>> [1] statmod_1.4.15 limma_3.12.1 >>>>>> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >>>>>> org.Hs.eg.db_2.7.1 >>>>>> >>>>>> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 >>>> KEGG.db_2.7.1 >>>>>> GO.db_2.7.1 AnnotationDbi_1.18.1 >>>>>> [11] affy_1.34.0 Biobase_2.16.0 >>>>>> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 >>> RSQLite_0.11.1 >>>>>> [16] DBI_0.2-5 oligo_1.20.4 >>>>>> oligoClasses_1.18.0 >>>>>> >>>>>> >>>>>> rmaOligoinvivo = oligo::rma(InVivodat1) >>>>>> Background correcting >>>>>> Normalizing >>>>>> Calculating Expression >>>>>> >>>>>>> rmaOligoinvitro = oligo::rma(InVitrodat1) >>>>>> Background correcting >>>>>> Normalizing >>>>>> Calculating Expression >>>>>> >>>>>>> maplot(rmaOligoinvivo) >>>>>>> maplot(rmaOligoinvitro) >>>>>>> InVivoTargets >>>>>> FileName Treatment >>>>>> 1 MC1 A >>>>>> 2 MC2 A >>>>>> 3 MC3 A >>>>>> 4 MC4 A >>>>>> 5 MC5 A >>>>>> 6 MC6 A >>>>>> 7 MC7 R >>>>>> 8 MC8 R >>>>>> 9 MC9 R >>>>>> 10 MC10 R >>>>>> 11 MC11 R >>>>>> 12 MC12 R >>>>>> 13 MC13 T >>>>>> 14 MC14 T >>>>>> 15 MC15 T >>>>>> 16 MC16 T >>>>>> 17 MC17 T >>>>>> 18 MC18 T >>>>>> >> InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") >>>>>>> InVitroTargets >>>>>> FileName Treatment Batch CD4 >>>>>> 1 MC19 RY 1 High >>>>>> 2 MC20 TY 1 Low >>>>>> 3 MC21 RY 2 High >>>>>> 4 MC22 TY 2 High >>>>>> 5 MC23 TY 2 Low >>>>>> 6 MC24 RY 2 High >>>>>> 7 MC25 TXY 1 Low >>>>>> 8 MC26 RXY 1 High >>>>>> 9 MC27 RXY 2 Low >>>>>> 10 MC28 TXY 2 High >>>>>> 11 MC29 RXY 2 High >>>>>> 12 MC30 TXY 2 High >>>>>> >>>>>> f.invivo<- factor(InVivoTargets$Treatment, levels = c("A", "R", >> "T")) >>>>>> design.invivo<- model.matrix(~0 + f.invivo) >>>>>> >>>>>>> colnames(design.invivo)<- c("A", "R", "T") >>>>>>> fit.invivo<- lmFit(rmaOligoinvivo, design.invivo) >>>>>>> contrast.matrix.invivo<- makeContrasts(R-A, T-R, T-A,levels = >>>>>> design.invivo) >>>>>> >>>>>>> fit2.invivo<- contrasts.fit(fit.invivo, contrast.matrix.invivo) >>>>>>> fit2.invivo<-eBayes(fit2.invivo) >>>>>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>>>> ID logFC AveExpr t P.Value adj.P.Val >>>>>> B >>>>>> >>>>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>> -2.023533 >>>>>> >>>>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>> -2.023533 >>>>>> >>>>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>> -2.023533 >>>>>> >>>>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>> -2.023533 >>>>>> >>>>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>>>>> -2.323922 >>>>>> >>>>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>>>>> -2.454618 >>>>>> >>>>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>>>>> -2.607991 >>>>>> >>>>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>>>>> -2.685960 >>>>>> >>>>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>>>>> -2.711203 >>>>>> >>>>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>>>>> -2.800385 >>>>>> I am progressing in a right way? Further I want to perform unpaired >> t >>>> test >>>>>> for comparing AvsT and so on. Any help will be really great. >>>>>> >>>>> Hi, Mitra. I think that looks about right. You have already >> performed >>>>> the unpaired t-test of AvsT (well, actually TvsA, but the p-values >> will >>>> be >>>>> the same) as coefficient 3. >>>>> >>>>> Sean >>>>> >>>>> >>>> >>>> >>>> -- >>>> Dr. Suparna Mitra >>>> Wolfson Centre for Personalised Medicine >>>> Department of Molecular and Clinical Pharmacology >>>> Institute of Translational Medicine University of Liverpool >>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>>> >>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>>> M: +44 (0) 7511387895 >>>> Email id: smitra at liverpool.ac.uk >>>> Alternative Email id: suparna.mitra.sm at gmail.com >>>> >>>> [[alternative HTML version deleted]] >>>> >>>> _______________________________________________ >>>> Bioconductor mailing list >>>> Bioconductor at r-project.org >>>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>>> Search the archives: >>>> http://news.gmane.org/gmane.science.biology.informatics.conductor >>>> >>> [[alternative HTML version deleted]] >>> >>> _______________________________________________ >>> Bioconductor mailing list >>> Bioconductor at r-project.org >>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>> Search the archives: >>> http://news.gmane.org/gmane.science.biology.informatics.conductor >>> >> >> >> -- >> Dr. Suparna Mitra >> Wolfson Centre for Personalised Medicine >> Department of Molecular and Clinical Pharmacology >> Institute of Translational Medicine University of Liverpool >> Block A: Waterhouse Buildings, L69 3GL Liverpool >> >> Tel. +44 (0)151 795 5394, Internal ext: 55394 >> M: +44 (0) 7511387895 >> Email id: smitra at liverpool.ac.uk >> Alternative Email id: suparna.mitra.sm at gmail.com >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at r-project.org >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >> > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor -- James W. MacDonald, M.S. Biostatistician University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Thanks Jim, I will try to do this. being a novice I am trying hard to find best possible analysis option. I know this experiment is complicated. Thanks for help. best wishes, Mitra On 7 September 2012 14:55, James W. MacDonald <jmacdon@uw.edu> wrote: > > > On 9/7/2012 7:26 AM, Sean Davis wrote: > >> On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra<smitra@liverpool.ac.uk>** >> wrote: >> >> Oh thanks.. I missed this point. But can you suggest me one more thing... >>> when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the same >>> result as adjust = "fdr". for topTable. Is it normal? >>> >>> Yes. They are the same. See the help for p.adjust for details. >> >> >> Further when I tried to do vennDiagram I was surprized to see 0 in all >>> circles. Thus I thought I must be doing something wrong. Sorry if my >>> question is silly. >>> >>> Unfortunately, you have no significantly differentially-expressed genes. >> Note that all of the adjusted p-values are pretty high. You can try to >> filter your genes based on variance before testing to try to reduce the >> number of genes entering your test and multiple correction. However, >> having worked with this kind of biological system (patients), you may >> suffering from a problem of a small biological effect in the setting of >> large biological variation. A larger sample size may be necessary. >> > > You may also be suffering from large technical variation, which could be > helped by applying array weights. See ?arrayWeights for more information. > > Best, > > Jim > > > >> Sean >> >> >> Here is what I tried. >>> >>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>> >>> ID logFC AveExpr t P.Value adj.P.Val >>> B >>> >>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>> -2.023533 >>> >>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>> -2.023533 >>> >>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>> -2.023533 >>> >>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>> -2.023533 >>> >>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>> -2.323922 >>> >>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>> -2.454618 >>> >>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>> -2.607991 >>> >>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>> -2.685960 >>> >>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>> -2.711203 >>> >>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>> -2.800385 >>> >>> topTable(fit2.invivo, coef = 2, adjust = "fdr") >>>> >>> ID logFC AveExpr t P.Value adj.P.Val >>> B >>> >>> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 >>> -2.469821 >>> >>> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 >>> -2.652426 >>> >>> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 >>> -2.662042 >>> >>> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 >>> -2.702782 >>> >>> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 >>> -2.707492 >>> >>> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 >>> -2.717698 >>> >>> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 >>> -2.734873 >>> >>> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 >>> -2.772143 >>> >>> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 >>> -2.779941 >>> >>> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 >>> -2.785214 >>> >>> topTable(fit2.invivo, coef = 3, adjust = "fdr") >>>> >>> ID logFC AveExpr t P.Value adj.P.Val >>> B >>> >>> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 >>> -0.6982314 >>> >>> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 >>> -1.2472882 >>> >>> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 >>> -1.2727646 >>> >>> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 >>> -1.2831553 >>> >>> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 >>> -1.4031177 >>> >>> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 >>> -1.4733549 >>> >>> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 >>> -1.5025187 >>> >>> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 >>> -1.5612836 >>> >>> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 >>> -1.6535466 >>> >>> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 >>> -1.6662493 >>> >>> results<- decideTests(fit2.invivo) >>>> vennDiagram(results) >>>> >>> see the plot attached. >>> Thanks, >>> Mitra >>> >>> >>> On 7 September 2012 12:03, Sean Davis<sdavis2@mail.nih.gov> wrote: >>> >>> On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra<smitra@liverpool.ac.uk>>>> >>>>> wrote: >>>>> Dear Sean, >>>>> I have been reading Bioconductor and limma user guide and thus this >>>>> >>>> is >>> >>>> I >>>> >>>>> tried. >>>>> But just being a novice, wanted to make sure if I am right. >>>>> I know I have perform t-test when I created the contrast, but can you >>>>> please help me how can I perform unpaired t-test here. I am concerned >>>>> >>>> as >>> >>>> the patients in groups are not same. >>>>> >>>>> >>>> The t-test you performed was unpaired; unpaired is the "default". >>>> >>>> Sean >>>> >>>> >>>> Thanks, >>>>> Mitra >>>>> >>>>> On 7 September 2012 11:41, Sean Davis<sdavis2@mail.nih.gov> wrote: >>>>> >>>>> >>>>>> On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra< >>>>>> >>>>> smitra@liverpool.ac.uk >>> >>>> wrote: >>>>>> >>>>>> Hello Group, >>>>>>> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using >>>>>>> >>>>>> BiC. >>> >>>> Previously i was using different softwares for this. And this is my >>>>>>> >>>>>> first >>>>> >>>>>> try with Bioconductor for big experiment. So thought to get some >>>>>>> >>>>>> advice >>>> >>>>> in >>>>> >>>>>> the beginning. >>>>>>> I have Three groups of patient: (In-vivo) >>>>>>> A-Acute reaction. Patient taking a drug X develops reaction. >>>>>>> R-recovered (6 weeks after acute reaction-not longer taking drug >>>>>>> >>>>>> X). >>> >>>> T-Tolerant. Patient on X and tolerating treatment. >>>>>>> >>>>>>> Now in in-vitro study we used another constant Y >>>>>>> RXY recovered and challenged with X+Y >>>>>>> RY recovered challenged with only Y. RXY vs RY are to exclude >>>>>>> >>>>>> effects >>>> >>>>> by >>>>>>> Y. >>>>>>> TXY tolerant and challenged with X+Y, >>>>>>> TY tolerant challenged with only Y. TXY vs TY are to exclude >>>>>>> >>>>>> effects >>> >>>> by >>>>> >>>>>> Y. >>>>>>> >>>>>>> No I want to check the cross relation and effects A vs R, RvsT and >>>>>>> >>>>>> Avs T >>>> >>>>> and differentially expressed genes for each comparison. And the >>>>>>> >>>>>> same >>> >>>> in >>>> >>>>> invitro. There are not same patients in different groups, thus I >>>>>>> >>>>>> think I >>>> >>>>> want to apply unpaired-t test. >>>>>>> >>>>>>> This is what I tried: >>>>>>> >>>>>>>> sessionInfo() >>>>>>>> >>>>>>> R version 2.15.1 (2012-06-22) >>>>>>> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >>>>>>> >>>>>>> locale: >>>>>>> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.**UTF-8/C/en_GB.UTF-8/en_GB.UTF-** >>>>>>> 8 >>>>>>> >>>>>>> attached base packages: >>>>>>> [1] stats graphics grDevices utils datasets methods base >>>>>>> >>>>>>> other attached packages: >>>>>>> [1] statmod_1.4.15 limma_3.12.1 >>>>>>> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >>>>>>> org.Hs.eg.db_2.7.1 >>>>>>> >>>>>>> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 >>>>>>> >>>>>> KEGG.db_2.7.1 >>>>> >>>>>> GO.db_2.7.1 AnnotationDbi_1.18.1 >>>>>>> [11] affy_1.34.0 Biobase_2.16.0 >>>>>>> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 >>>>>>> >>>>>> RSQLite_0.11.1 >>>> >>>>> [16] DBI_0.2-5 oligo_1.20.4 >>>>>>> oligoClasses_1.18.0 >>>>>>> >>>>>>> >>>>>>> rmaOligoinvivo = oligo::rma(InVivodat1) >>>>>>> Background correcting >>>>>>> Normalizing >>>>>>> Calculating Expression >>>>>>> >>>>>>> rmaOligoinvitro = oligo::rma(InVitrodat1) >>>>>>>> >>>>>>> Background correcting >>>>>>> Normalizing >>>>>>> Calculating Expression >>>>>>> >>>>>>> maplot(rmaOligoinvivo) >>>>>>>> maplot(rmaOligoinvitro) >>>>>>>> InVivoTargets >>>>>>>> >>>>>>> FileName Treatment >>>>>>> 1 MC1 A >>>>>>> 2 MC2 A >>>>>>> 3 MC3 A >>>>>>> 4 MC4 A >>>>>>> 5 MC5 A >>>>>>> 6 MC6 A >>>>>>> 7 MC7 R >>>>>>> 8 MC8 R >>>>>>> 9 MC9 R >>>>>>> 10 MC10 R >>>>>>> 11 MC11 R >>>>>>> 12 MC12 R >>>>>>> 13 MC13 T >>>>>>> 14 MC14 T >>>>>>> 15 MC15 T >>>>>>> 16 MC16 T >>>>>>> 17 MC17 T >>>>>>> 18 MC18 T >>>>>>> >>>>>>> InVitroTargets=readTargets("~/**Desktop/Recent/Liverpool- work-** >>> related/Micro_RawData/**InVitroTargets.txt") >>> >>>> InVitroTargets >>>>>>>> >>>>>>> FileName Treatment Batch CD4 >>>>>>> 1 MC19 RY 1 High >>>>>>> 2 MC20 TY 1 Low >>>>>>> 3 MC21 RY 2 High >>>>>>> 4 MC22 TY 2 High >>>>>>> 5 MC23 TY 2 Low >>>>>>> 6 MC24 RY 2 High >>>>>>> 7 MC25 TXY 1 Low >>>>>>> 8 MC26 RXY 1 High >>>>>>> 9 MC27 RXY 2 Low >>>>>>> 10 MC28 TXY 2 High >>>>>>> 11 MC29 RXY 2 High >>>>>>> 12 MC30 TXY 2 High >>>>>>> >>>>>>> f.invivo<- factor(InVivoTargets$**Treatment, levels = c("A", "R", >>>>>>> >>>>>> "T")) >>> >>>> design.invivo<- model.matrix(~0 + f.invivo) >>>>>>> >>>>>>> colnames(design.invivo)<- c("A", "R", "T") >>>>>>>> fit.invivo<- lmFit(rmaOligoinvivo, design.invivo) >>>>>>>> contrast.matrix.invivo<- makeContrasts(R-A, T-R, T-A,levels = >>>>>>>> >>>>>>> design.invivo) >>>>>>> >>>>>>> fit2.invivo<- contrasts.fit(fit.invivo, contrast.matrix.invivo) >>>>>>>> fit2.invivo<-eBayes(fit2.**invivo) >>>>>>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>>>>>> >>>>>>> ID logFC AveExpr t P.Value adj.P.Val >>>>>>> B >>>>>>> >>>>>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>> -2.023533 >>>>>>> >>>>>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>> -2.023533 >>>>>>> >>>>>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>> -2.023533 >>>>>>> >>>>>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>> -2.023533 >>>>>>> >>>>>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>>>>>> -2.323922 >>>>>>> >>>>>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>>>>>> -2.454618 >>>>>>> >>>>>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>>>>>> -2.607991 >>>>>>> >>>>>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>>>>>> -2.685960 >>>>>>> >>>>>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>>>>>> -2.711203 >>>>>>> >>>>>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>>>>>> -2.800385 >>>>>>> I am progressing in a right way? Further I want to perform unpaired >>>>>>> >>>>>> t >>> >>>> test >>>>> >>>>>> for comparing AvsT and so on. Any help will be really great. >>>>>>> >>>>>>> Hi, Mitra. I think that looks about right. You have already >>>>>> >>>>> performed >>> >>>> the unpaired t-test of AvsT (well, actually TvsA, but the p-values >>>>>> >>>>> will >>> >>>> be >>>>> >>>>>> the same) as coefficient 3. >>>>>> >>>>>> Sean >>>>>> >>>>>> >>>>>> >>>>> >>>>> -- >>>>> Dr. Suparna Mitra >>>>> Wolfson Centre for Personalised Medicine >>>>> Department of Molecular and Clinical Pharmacology >>>>> Institute of Translational Medicine University of Liverpool >>>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>>>> >>>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>>>> M: +44 (0) 7511387895 >>>>> Email id: smitra@liverpool.ac.uk >>>>> Alternative Email id: suparna.mitra.sm@gmail.com >>>>> >>>>> [[alternative HTML version deleted]] >>>>> >>>>> ______________________________**_________________ >>>>> Bioconductor mailing list >>>>> Bioconductor@r-project.org >>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: sta="" t.ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>>> Search the archives: >>>>> http://news.gmane.org/gmane.**science.biology.informatics.**cond uctor<http: news.gmane.org="" gmane.science.biology.informatics.conducto="" r=""> >>>>> >>>>> [[alternative HTML version deleted]] >>>> >>>> ______________________________**_________________ >>>> Bioconductor mailing list >>>> Bioconductor@r-project.org >>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat="" .ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>> Search the archives: >>>> http://news.gmane.org/gmane.**science.biology.informatics.**condu ctor<http: news.gmane.org="" gmane.science.biology.informatics.conductor=""> >>>> >>>> >>> >>> -- >>> Dr. Suparna Mitra >>> Wolfson Centre for Personalised Medicine >>> Department of Molecular and Clinical Pharmacology >>> Institute of Translational Medicine University of Liverpool >>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>> >>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>> M: +44 (0) 7511387895 >>> Email id: smitra@liverpool.ac.uk >>> Alternative Email id: suparna.mitra.sm@gmail.com >>> >>> ______________________________**_________________ >>> Bioconductor mailing list >>> Bioconductor@r-project.org >>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat.="" ethz.ch="" mailman="" listinfo="" bioconductor=""> >>> Search the archives: >>> http://news.gmane.org/gmane.**science.biology.informatics.**conduc tor<http: news.gmane.org="" gmane.science.biology.informatics.conductor=""> >>> >>> [[alternative HTML version deleted]] >> >> ______________________________**_________________ >> Bioconductor mailing list >> Bioconductor@r-project.org >> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat.e="" thz.ch="" mailman="" listinfo="" bioconductor=""> >> Search the archives: http://news.gmane.org/gmane.** >> science.biology.informatics.**conductor<http: news.gmane.org="" gmane="" .science.biology.informatics.conductor=""> >> > > -- > James W. MacDonald, M.S. > Biostatistician > University of Washington > Environmental and Occupational Health Sciences > 4225 Roosevelt Way NE, # 100 > Seattle WA 98105-6099 > > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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Hello Jim, Sean, For your previous suggestion. I am trying to do accordingly. Can you please suggest how I can do the following to match a previous set of experiment. #Entitylist : A v R Filtered on Error - CV < 300.0 percent #Interpretation : A vs R Treatment #Experiment: RMA #p-value cut-off:0.05 #Selected Test : T Test unpaired #p-value computation: Asymptotic Until now I have done rma on direct data and performed t-test. But how can I 'Filtered on Error - CV' and select p-value computation as 'Asymptotic'? Thanks a lot, Mitra > > On 7 September 2012 14:55, James W. MacDonald <jmacdon@uw.edu> wrote: > >> >> >> On 9/7/2012 7:26 AM, Sean Davis wrote: >> >>> On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra<smitra@liverpool.ac.uk>** >>> wrote: >>> >>> Oh thanks.. I missed this point. But can you suggest me one more >>>> thing... >>>> when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the same >>>> result as adjust = "fdr". for topTable. Is it normal? >>>> >>>> Yes. They are the same. See the help for p.adjust for details. >>> >>> >>> Further when I tried to do vennDiagram I was surprized to see 0 in all >>>> circles. Thus I thought I must be doing something wrong. Sorry if my >>>> question is silly. >>>> >>>> Unfortunately, you have no significantly differentially- expressed >>> genes. >>> Note that all of the adjusted p-values are pretty high. You can try to >>> filter your genes based on variance before testing to try to reduce the >>> number of genes entering your test and multiple correction. However, >>> having worked with this kind of biological system (patients), you may >>> suffering from a problem of a small biological effect in the setting of >>> large biological variation. A larger sample size may be necessary. >>> >> >> You may also be suffering from large technical variation, which could be >> helped by applying array weights. See ?arrayWeights for more information. >> >> Best, >> >> Jim >> >> >> >>> Sean >>> >>> >>> Here is what I tried. >>>> >>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>>> >>>> ID logFC AveExpr t P.Value adj.P.Val >>>> B >>>> >>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>>> -2.323922 >>>> >>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>>> -2.454618 >>>> >>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>>> -2.607991 >>>> >>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>>> -2.685960 >>>> >>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>>> -2.711203 >>>> >>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>>> -2.800385 >>>> >>>> topTable(fit2.invivo, coef = 2, adjust = "fdr") >>>>> >>>> ID logFC AveExpr t P.Value adj.P.Val >>>> B >>>> >>>> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 >>>> -2.469821 >>>> >>>> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 >>>> -2.652426 >>>> >>>> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 >>>> -2.662042 >>>> >>>> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 >>>> -2.702782 >>>> >>>> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 >>>> -2.707492 >>>> >>>> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 >>>> -2.717698 >>>> >>>> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 >>>> -2.734873 >>>> >>>> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 >>>> -2.772143 >>>> >>>> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 >>>> -2.779941 >>>> >>>> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 >>>> -2.785214 >>>> >>>> topTable(fit2.invivo, coef = 3, adjust = "fdr") >>>>> >>>> ID logFC AveExpr t P.Value adj.P.Val >>>> B >>>> >>>> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 >>>> -0.6982314 >>>> >>>> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 >>>> -1.2472882 >>>> >>>> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 >>>> -1.2727646 >>>> >>>> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 >>>> -1.2831553 >>>> >>>> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 >>>> -1.4031177 >>>> >>>> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 >>>> -1.4733549 >>>> >>>> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 >>>> -1.5025187 >>>> >>>> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 >>>> -1.5612836 >>>> >>>> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 >>>> -1.6535466 >>>> >>>> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 >>>> -1.6662493 >>>> >>>> results<- decideTests(fit2.invivo) >>>>> vennDiagram(results) >>>>> >>>> see the plot attached. >>>> Thanks, >>>> Mitra >>>> >>>> >>>> On 7 September 2012 12:03, Sean Davis<sdavis2@mail.nih.gov> wrote: >>>> >>>> On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra<smitra@liverpool.ac.uk>>>>> >>>>>> wrote: >>>>>> Dear Sean, >>>>>> I have been reading Bioconductor and limma user guide and thus this >>>>>> >>>>> is >>>> >>>>> I >>>>> >>>>>> tried. >>>>>> But just being a novice, wanted to make sure if I am right. >>>>>> I know I have perform t-test when I created the contrast, but can you >>>>>> please help me how can I perform unpaired t-test here. I am concerned >>>>>> >>>>> as >>>> >>>>> the patients in groups are not same. >>>>>> >>>>>> >>>>> The t-test you performed was unpaired; unpaired is the "default". >>>>> >>>>> Sean >>>>> >>>>> >>>>> Thanks, >>>>>> Mitra >>>>>> >>>>>> On 7 September 2012 11:41, Sean Davis<sdavis2@mail.nih.gov> wrote: >>>>>> >>>>>> >>>>>>> On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra< >>>>>>> >>>>>> smitra@liverpool.ac.uk >>>> >>>>> wrote: >>>>>>> >>>>>>> Hello Group, >>>>>>>> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using >>>>>>>> >>>>>>> BiC. >>>> >>>>> Previously i was using different softwares for this. And this is my >>>>>>>> >>>>>>> first >>>>>> >>>>>>> try with Bioconductor for big experiment. So thought to get some >>>>>>>> >>>>>>> advice >>>>> >>>>>> in >>>>>> >>>>>>> the beginning. >>>>>>>> I have Three groups of patient: (In-vivo) >>>>>>>> A-Acute reaction. Patient taking a drug X develops reaction. >>>>>>>> R-recovered (6 weeks after acute reaction-not longer taking drug >>>>>>>> >>>>>>> X). >>>> >>>>> T-Tolerant. Patient on X and tolerating treatment. >>>>>>>> >>>>>>>> Now in in-vitro study we used another constant Y >>>>>>>> RXY recovered and challenged with X+Y >>>>>>>> RY recovered challenged with only Y. RXY vs RY are to exclude >>>>>>>> >>>>>>> effects >>>>> >>>>>> by >>>>>>>> Y. >>>>>>>> TXY tolerant and challenged with X+Y, >>>>>>>> TY tolerant challenged with only Y. TXY vs TY are to exclude >>>>>>>> >>>>>>> effects >>>> >>>>> by >>>>>> >>>>>>> Y. >>>>>>>> >>>>>>>> No I want to check the cross relation and effects A vs R, RvsT and >>>>>>>> >>>>>>> Avs T >>>>> >>>>>> and differentially expressed genes for each comparison. And the >>>>>>>> >>>>>>> same >>>> >>>>> in >>>>> >>>>>> invitro. There are not same patients in different groups, thus I >>>>>>>> >>>>>>> think I >>>>> >>>>>> want to apply unpaired-t test. >>>>>>>> >>>>>>>> This is what I tried: >>>>>>>> >>>>>>>>> sessionInfo() >>>>>>>>> >>>>>>>> R version 2.15.1 (2012-06-22) >>>>>>>> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >>>>>>>> >>>>>>>> locale: >>>>>>>> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.**UTF-8/C/en_GB.UTF-8/en_GB.UTF-* >>>>>>>> *8 >>>>>>>> >>>>>>>> attached base packages: >>>>>>>> [1] stats graphics grDevices utils datasets methods base >>>>>>>> >>>>>>>> other attached packages: >>>>>>>> [1] statmod_1.4.15 limma_3.12.1 >>>>>>>> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >>>>>>>> org.Hs.eg.db_2.7.1 >>>>>>>> >>>>>>>> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 >>>>>>>> >>>>>>> KEGG.db_2.7.1 >>>>>> >>>>>>> GO.db_2.7.1 AnnotationDbi_1.18.1 >>>>>>>> [11] affy_1.34.0 Biobase_2.16.0 >>>>>>>> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 >>>>>>>> >>>>>>> RSQLite_0.11.1 >>>>> >>>>>> [16] DBI_0.2-5 oligo_1.20.4 >>>>>>>> oligoClasses_1.18.0 >>>>>>>> >>>>>>>> >>>>>>>> rmaOligoinvivo = oligo::rma(InVivodat1) >>>>>>>> Background correcting >>>>>>>> Normalizing >>>>>>>> Calculating Expression >>>>>>>> >>>>>>>> rmaOligoinvitro = oligo::rma(InVitrodat1) >>>>>>>>> >>>>>>>> Background correcting >>>>>>>> Normalizing >>>>>>>> Calculating Expression >>>>>>>> >>>>>>>> maplot(rmaOligoinvivo) >>>>>>>>> maplot(rmaOligoinvitro) >>>>>>>>> InVivoTargets >>>>>>>>> >>>>>>>> FileName Treatment >>>>>>>> 1 MC1 A >>>>>>>> 2 MC2 A >>>>>>>> 3 MC3 A >>>>>>>> 4 MC4 A >>>>>>>> 5 MC5 A >>>>>>>> 6 MC6 A >>>>>>>> 7 MC7 R >>>>>>>> 8 MC8 R >>>>>>>> 9 MC9 R >>>>>>>> 10 MC10 R >>>>>>>> 11 MC11 R >>>>>>>> 12 MC12 R >>>>>>>> 13 MC13 T >>>>>>>> 14 MC14 T >>>>>>>> 15 MC15 T >>>>>>>> 16 MC16 T >>>>>>>> 17 MC17 T >>>>>>>> 18 MC18 T >>>>>>>> >>>>>>>> InVitroTargets=readTargets("~/**Desktop/Recent/Liverpool- work-** >>>> related/Micro_RawData/**InVitroTargets.txt") >>>> >>>>> InVitroTargets >>>>>>>>> >>>>>>>> FileName Treatment Batch CD4 >>>>>>>> 1 MC19 RY 1 High >>>>>>>> 2 MC20 TY 1 Low >>>>>>>> 3 MC21 RY 2 High >>>>>>>> 4 MC22 TY 2 High >>>>>>>> 5 MC23 TY 2 Low >>>>>>>> 6 MC24 RY 2 High >>>>>>>> 7 MC25 TXY 1 Low >>>>>>>> 8 MC26 RXY 1 High >>>>>>>> 9 MC27 RXY 2 Low >>>>>>>> 10 MC28 TXY 2 High >>>>>>>> 11 MC29 RXY 2 High >>>>>>>> 12 MC30 TXY 2 High >>>>>>>> >>>>>>>> f.invivo<- factor(InVivoTargets$**Treatment, levels = c("A", "R", >>>>>>>> >>>>>>> "T")) >>>> >>>>> design.invivo<- model.matrix(~0 + f.invivo) >>>>>>>> >>>>>>>> colnames(design.invivo)<- c("A", "R", "T") >>>>>>>>> fit.invivo<- lmFit(rmaOligoinvivo, design.invivo) >>>>>>>>> contrast.matrix.invivo<- makeContrasts(R-A, T-R, T-A,levels = >>>>>>>>> >>>>>>>> design.invivo) >>>>>>>> >>>>>>>> fit2.invivo<- contrasts.fit(fit.invivo, contrast.matrix.invivo) >>>>>>>>> fit2.invivo<-eBayes(fit2.**invivo) >>>>>>>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>>>>>>> >>>>>>>> ID logFC AveExpr t P.Value adj.P.Val >>>>>>>> B >>>>>>>> >>>>>>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>>>>>>> -2.323922 >>>>>>>> >>>>>>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>>>>>>> -2.454618 >>>>>>>> >>>>>>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>>>>>>> -2.607991 >>>>>>>> >>>>>>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>>>>>>> -2.685960 >>>>>>>> >>>>>>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>>>>>>> -2.711203 >>>>>>>> >>>>>>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>>>>>>> -2.800385 >>>>>>>> I am progressing in a right way? Further I want to perform unpaired >>>>>>>> >>>>>>> t >>>> >>>>> test >>>>>> >>>>>>> for comparing AvsT and so on. Any help will be really great. >>>>>>>> >>>>>>>> Hi, Mitra. I think that looks about right. You have already >>>>>>> >>>>>> performed >>>> >>>>> the unpaired t-test of AvsT (well, actually TvsA, but the p-values >>>>>>> >>>>>> will >>>> >>>>> be >>>>>> >>>>>>> the same) as coefficient 3. >>>>>>> >>>>>>> Sean >>>>>>> >>>>>>> >>>>>>> >>>>>> >>>>>> -- >>>>>> Dr. Suparna Mitra >>>>>> Wolfson Centre for Personalised Medicine >>>>>> Department of Molecular and Clinical Pharmacology >>>>>> Institute of Translational Medicine University of Liverpool >>>>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>>>>> >>>>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>>>>> M: +44 (0) 7511387895 >>>>>> Email id: smitra@liverpool.ac.uk >>>>>> Alternative Email id: suparna.mitra.sm@gmail.com >>>>>> >>>>>> [[alternative HTML version deleted]] >>>>>> >>>>>> ______________________________**_________________ >>>>>> Bioconductor mailing list >>>>>> Bioconductor@r-project.org >>>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: st="" at.ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>>>> Search the archives: >>>>>> http://news.gmane.org/gmane.**science.biology.informatics.**con ductor<http: news.gmane.org="" gmane.science.biology.informatics.conduct="" or=""> >>>>>> >>>>>> [[alternative HTML version deleted]] >>>>> >>>>> ______________________________**_________________ >>>>> Bioconductor mailing list >>>>> Bioconductor@r-project.org >>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: sta="" t.ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>>> Search the archives: >>>>> http://news.gmane.org/gmane.**science.biology.informatics.**cond uctor<http: news.gmane.org="" gmane.science.biology.informatics.conducto="" r=""> >>>>> >>>>> >>>> >>>> -- >>>> Dr. Suparna Mitra >>>> Wolfson Centre for Personalised Medicine >>>> Department of Molecular and Clinical Pharmacology >>>> Institute of Translational Medicine University of Liverpool >>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>>> >>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>>> M: +44 (0) 7511387895 >>>> Email id: smitra@liverpool.ac.uk >>>> Alternative Email id: suparna.mitra.sm@gmail.com >>>> >>>> ______________________________**_________________ >>>> Bioconductor mailing list >>>> Bioconductor@r-project.org >>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat="" .ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>> Search the archives: >>>> http://news.gmane.org/gmane.**science.biology.informatics.**condu ctor<http: news.gmane.org="" gmane.science.biology.informatics.conductor=""> >>>> >>>> [[alternative HTML version deleted]] >>> >>> ______________________________**_________________ >>> Bioconductor mailing list >>> Bioconductor@r-project.org >>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat.="" ethz.ch="" mailman="" listinfo="" bioconductor=""> >>> Search the archives: http://news.gmane.org/gmane.** >>> science.biology.informatics.**conductor<http: news.gmane.org="" gman="" e.science.biology.informatics.conductor=""> >>> >> >> -- >> James W. MacDonald, M.S. >> Biostatistician >> University of Washington >> Environmental and Occupational Health Sciences >> 4225 Roosevelt Way NE, # 100 >> Seattle WA 98105-6099 >> >> > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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Hello group, Can anybody please you please suggest how I can do the following in BioC to match a previous set of experiment. #Entitylist : A v R Filtered on Error - CV < 300.0 percent #Interpretation : A vs R Treatment #Experiment: RMA #p-value cut-off:0.05 #Selected Test : T Test unpaired #p-value computation: Asymptotic Until now I have done rma on direct data and performed t-test. But how can I 'Filtered on Error - CV' and select p-value computation as 'Asymptotic'? This is what I did. > sessionInfo() R version 2.15.1 (2012-06-22) Platform: i386-apple-darwin9.8.0/i386 (32-bit) locale: [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] statmod_1.4.15 limma_3.12.1 annotate_1.34.1 hugene10stprobeset.db_8.0.1 org.Hs.eg.db_2.7.1 [6] BiocInstaller_1.4.7 affycoretools_1.28.0 KEGG.db_2.7.1 GO.db_2.7.1 AnnotationDbi_1.18.1 [11] affy_1.34.0 Biobase_2.16.0 BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 [16] DBI_0.2-5 oligo_1.20.4 oligoClasses_1.18.0 rmaOligoinvivo = oligo::rma(InVivodat1) Background correcting Normalizing Calculating Expression > rmaOligoinvitro = oligo::rma(InVitrodat1) Background correcting Normalizing Calculating Expression > maplot(rmaOligoinvivo) > maplot(rmaOligoinvitro) > InVivoTargets FileName Treatment 1 MC1 A 2 MC2 A 3 MC3 A 4 MC4 A 5 MC5 A 6 MC6 A 7 MC7 R 8 MC8 R 9 MC9 R 10 MC10 R 11 MC11 R 12 MC12 R 13 MC13 T 14 MC14 T 15 MC15 T 16 MC16 T 17 MC17 T 18 MC18 T > InVitroTargets FileName Treatment Batch CD4 1 MC19 RY 1 High 2 MC20 TY 1 Low 3 MC21 RY 2 High 4 MC22 TY 2 High 5 MC23 TY 2 Low 6 MC24 RY 2 High 7 MC25 TXY 1 Low 8 MC26 RXY 1 High 9 MC27 RXY 2 Low 10 MC28 TXY 2 High11 MC29 RXY 2 High 12 MC30 TXY 2 High f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) design.invivo <- model.matrix(~0 + f.invivo) > > colnames(design.invivo) <- c("A", "R", "T") > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = design.invivo) > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > fit2.invivo <-eBayes(fit2.invivo) > topTable(fit2.invivo, coef = 1, adjust = "fdr") ID logFC AveExpr t P.Value adj.P.Val B 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 -2.023533 ... Thanks a lot, Mitra > > On 7 September 2012 14:55, James W. MacDonald <jmacdon@uw.edu> wrote: > >> >> >> On 9/7/2012 7:26 AM, Sean Davis wrote: >> >>> On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra<smitra@liverpool.ac.uk>** >>> wrote: >>> >>> Oh thanks.. I missed this point. But can you suggest me one more >>>> thing... >>>> when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the same >>>> result as adjust = "fdr". for topTable. Is it normal? >>>> >>>> Yes. They are the same. See the help for p.adjust for details. >>> >>> >>> Further when I tried to do vennDiagram I was surprized to see 0 in all >>>> circles. Thus I thought I must be doing something wrong. Sorry if my >>>> question is silly. >>>> >>>> Unfortunately, you have no significantly differentially- expressed >>> genes. >>> Note that all of the adjusted p-values are pretty high. You can try to >>> filter your genes based on variance before testing to try to reduce the >>> number of genes entering your test and multiple correction. However, >>> having worked with this kind of biological system (patients), you may >>> suffering from a problem of a small biological effect in the setting of >>> large biological variation. A larger sample size may be necessary. >>> >> >> You may also be suffering from large technical variation, which could be >> helped by applying array weights. See ?arrayWeights for more information. >> >> Best, >> >> Jim >> >> >> >>> Sean >>> >>> >>> Here is what I tried. >>>> >>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>>> >>>> ID logFC AveExpr t P.Value adj.P.Val >>>> B >>>> >>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>> -2.023533 >>>> >>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>>> -2.323922 >>>> >>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>>> -2.454618 >>>> >>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>>> -2.607991 >>>> >>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>>> -2.685960 >>>> >>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>>> -2.711203 >>>> >>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>>> -2.800385 >>>> >>>> topTable(fit2.invivo, coef = 2, adjust = "fdr") >>>>> >>>> ID logFC AveExpr t P.Value adj.P.Val >>>> B >>>> >>>> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 >>>> -2.469821 >>>> >>>> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 >>>> -2.652426 >>>> >>>> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 >>>> -2.662042 >>>> >>>> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 >>>> -2.702782 >>>> >>>> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 >>>> -2.707492 >>>> >>>> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 >>>> -2.717698 >>>> >>>> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 >>>> -2.734873 >>>> >>>> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 >>>> -2.772143 >>>> >>>> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 >>>> -2.779941 >>>> >>>> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 >>>> -2.785214 >>>> >>>> topTable(fit2.invivo, coef = 3, adjust = "fdr") >>>>> >>>> ID logFC AveExpr t P.Value adj.P.Val >>>> B >>>> >>>> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 >>>> -0.6982314 >>>> >>>> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 >>>> -1.2472882 >>>> >>>> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 >>>> -1.2727646 >>>> >>>> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 >>>> -1.2831553 >>>> >>>> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 >>>> -1.4031177 >>>> >>>> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 >>>> -1.4733549 >>>> >>>> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 >>>> -1.5025187 >>>> >>>> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 >>>> -1.5612836 >>>> >>>> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 >>>> -1.6535466 >>>> >>>> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 >>>> -1.6662493 >>>> >>>> results<- decideTests(fit2.invivo) >>>>> vennDiagram(results) >>>>> >>>> see the plot attached. >>>> Thanks, >>>> Mitra >>>> >>>> >>>> On 7 September 2012 12:03, Sean Davis<sdavis2@mail.nih.gov> wrote: >>>> >>>> On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra<smitra@liverpool.ac.uk>>>>> >>>>>> wrote: >>>>>> Dear Sean, >>>>>> I have been reading Bioconductor and limma user guide and thus this >>>>>> >>>>> is >>>> >>>>> I >>>>> >>>>>> tried. >>>>>> But just being a novice, wanted to make sure if I am right. >>>>>> I know I have perform t-test when I created the contrast, but can you >>>>>> please help me how can I perform unpaired t-test here. I am concerned >>>>>> >>>>> as >>>> >>>>> the patients in groups are not same. >>>>>> >>>>>> >>>>> The t-test you performed was unpaired; unpaired is the "default". >>>>> >>>>> Sean >>>>> >>>>> >>>>> Thanks, >>>>>> Mitra >>>>>> >>>>>> On 7 September 2012 11:41, Sean Davis<sdavis2@mail.nih.gov> wrote: >>>>>> >>>>>> >>>>>>> On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra< >>>>>>> >>>>>> smitra@liverpool.ac.uk >>>> >>>>> wrote: >>>>>>> >>>>>>> Hello Group, >>>>>>>> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using >>>>>>>> >>>>>>> BiC. >>>> >>>>> Previously i was using different softwares for this. And this is my >>>>>>>> >>>>>>> first >>>>>> >>>>>>> try with Bioconductor for big experiment. So thought to get some >>>>>>>> >>>>>>> advice >>>>> >>>>>> in >>>>>> >>>>>>> the beginning. >>>>>>>> I have Three groups of patient: (In-vivo) >>>>>>>> A-Acute reaction. Patient taking a drug X develops reaction. >>>>>>>> R-recovered (6 weeks after acute reaction-not longer taking drug >>>>>>>> >>>>>>> X). >>>> >>>>> T-Tolerant. Patient on X and tolerating treatment. >>>>>>>> >>>>>>>> Now in in-vitro study we used another constant Y >>>>>>>> RXY recovered and challenged with X+Y >>>>>>>> RY recovered challenged with only Y. RXY vs RY are to exclude >>>>>>>> >>>>>>> effects >>>>> >>>>>> by >>>>>>>> Y. >>>>>>>> TXY tolerant and challenged with X+Y, >>>>>>>> TY tolerant challenged with only Y. TXY vs TY are to exclude >>>>>>>> >>>>>>> effects >>>> >>>>> by >>>>>> >>>>>>> Y. >>>>>>>> >>>>>>>> No I want to check the cross relation and effects A vs R, RvsT and >>>>>>>> >>>>>>> Avs T >>>>> >>>>>> and differentially expressed genes for each comparison. And the >>>>>>>> >>>>>>> same >>>> >>>>> in >>>>> >>>>>> invitro. There are not same patients in different groups, thus I >>>>>>>> >>>>>>> think I >>>>> >>>>>> want to apply unpaired-t test. >>>>>>>> >>>>>>>> This is what I tried: >>>>>>>> >>>>>>>>> sessionInfo() >>>>>>>>> >>>>>>>> R version 2.15.1 (2012-06-22) >>>>>>>> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >>>>>>>> >>>>>>>> locale: >>>>>>>> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.**UTF-8/C/en_GB.UTF-8/en_GB.UTF-* >>>>>>>> *8 >>>>>>>> >>>>>>>> attached base packages: >>>>>>>> [1] stats graphics grDevices utils datasets methods base >>>>>>>> >>>>>>>> other attached packages: >>>>>>>> [1] statmod_1.4.15 limma_3.12.1 >>>>>>>> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >>>>>>>> org.Hs.eg.db_2.7.1 >>>>>>>> >>>>>>>> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 >>>>>>>> >>>>>>> KEGG.db_2.7.1 >>>>>> >>>>>>> GO.db_2.7.1 AnnotationDbi_1.18.1 >>>>>>>> [11] affy_1.34.0 Biobase_2.16.0 >>>>>>>> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 >>>>>>>> >>>>>>> RSQLite_0.11.1 >>>>> >>>>>> [16] DBI_0.2-5 oligo_1.20.4 >>>>>>>> oligoClasses_1.18.0 >>>>>>>> >>>>>>>> >>>>>>>> rmaOligoinvivo = oligo::rma(InVivodat1) >>>>>>>> Background correcting >>>>>>>> Normalizing >>>>>>>> Calculating Expression >>>>>>>> >>>>>>>> rmaOligoinvitro = oligo::rma(InVitrodat1) >>>>>>>>> >>>>>>>> Background correcting >>>>>>>> Normalizing >>>>>>>> Calculating Expression >>>>>>>> >>>>>>>> maplot(rmaOligoinvivo) >>>>>>>>> maplot(rmaOligoinvitro) >>>>>>>>> InVivoTargets >>>>>>>>> >>>>>>>> FileName Treatment >>>>>>>> 1 MC1 A >>>>>>>> 2 MC2 A >>>>>>>> 3 MC3 A >>>>>>>> 4 MC4 A >>>>>>>> 5 MC5 A >>>>>>>> 6 MC6 A >>>>>>>> 7 MC7 R >>>>>>>> 8 MC8 R >>>>>>>> 9 MC9 R >>>>>>>> 10 MC10 R >>>>>>>> 11 MC11 R >>>>>>>> 12 MC12 R >>>>>>>> 13 MC13 T >>>>>>>> 14 MC14 T >>>>>>>> 15 MC15 T >>>>>>>> 16 MC16 T >>>>>>>> 17 MC17 T >>>>>>>> 18 MC18 T >>>>>>>> >>>>>>>> InVitroTargets=readTargets("~/**Desktop/Recent/Liverpool- work-** >>>> related/Micro_RawData/**InVitroTargets.txt") >>>> >>>>> InVitroTargets >>>>>>>>> >>>>>>>> FileName Treatment Batch CD4 >>>>>>>> 1 MC19 RY 1 High >>>>>>>> 2 MC20 TY 1 Low >>>>>>>> 3 MC21 RY 2 High >>>>>>>> 4 MC22 TY 2 High >>>>>>>> 5 MC23 TY 2 Low >>>>>>>> 6 MC24 RY 2 High >>>>>>>> 7 MC25 TXY 1 Low >>>>>>>> 8 MC26 RXY 1 High >>>>>>>> 9 MC27 RXY 2 Low >>>>>>>> 10 MC28 TXY 2 High >>>>>>>> 11 MC29 RXY 2 High >>>>>>>> 12 MC30 TXY 2 High >>>>>>>> >>>>>>>> f.invivo<- factor(InVivoTargets$**Treatment, levels = c("A", "R", >>>>>>>> >>>>>>> "T")) >>>> >>>>> design.invivo<- model.matrix(~0 + f.invivo) >>>>>>>> >>>>>>>> colnames(design.invivo)<- c("A", "R", "T") >>>>>>>>> fit.invivo<- lmFit(rmaOligoinvivo, design.invivo) >>>>>>>>> contrast.matrix.invivo<- makeContrasts(R-A, T-R, T-A,levels = >>>>>>>>> >>>>>>>> design.invivo) >>>>>>>> >>>>>>>> fit2.invivo<- contrasts.fit(fit.invivo, contrast.matrix.invivo) >>>>>>>>> fit2.invivo<-eBayes(fit2.**invivo) >>>>>>>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >>>>>>>>> >>>>>>>> ID logFC AveExpr t P.Value adj.P.Val >>>>>>>> B >>>>>>>> >>>>>>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >>>>>>>> -2.023533 >>>>>>>> >>>>>>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >>>>>>>> -2.323922 >>>>>>>> >>>>>>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >>>>>>>> -2.454618 >>>>>>>> >>>>>>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >>>>>>>> -2.607991 >>>>>>>> >>>>>>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >>>>>>>> -2.685960 >>>>>>>> >>>>>>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >>>>>>>> -2.711203 >>>>>>>> >>>>>>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >>>>>>>> -2.800385 >>>>>>>> I am progressing in a right way? Further I want to perform unpaired >>>>>>>> >>>>>>> t >>>> >>>>> test >>>>>> >>>>>>> for comparing AvsT and so on. Any help will be really great. >>>>>>>> >>>>>>>> Hi, Mitra. I think that looks about right. You have already >>>>>>> >>>>>> performed >>>> >>>>> the unpaired t-test of AvsT (well, actually TvsA, but the p-values >>>>>>> >>>>>> will >>>> >>>>> be >>>>>> >>>>>>> the same) as coefficient 3. >>>>>>> >>>>>>> Sean >>>>>>> >>>>>>> >>>>>>> >>>>>> >>>>>> -- >>>>>> Dr. Suparna Mitra >>>>>> Wolfson Centre for Personalised Medicine >>>>>> Department of Molecular and Clinical Pharmacology >>>>>> Institute of Translational Medicine University of Liverpool >>>>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>>>>> >>>>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>>>>> M: +44 (0) 7511387895 >>>>>> Email id: smitra@liverpool.ac.uk >>>>>> Alternative Email id: suparna.mitra.sm@gmail.com >>>>>> >>>>>> [[alternative HTML version deleted]] >>>>>> >>>>>> ______________________________**_________________ >>>>>> Bioconductor mailing list >>>>>> Bioconductor@r-project.org >>>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: st="" at.ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>>>> Search the archives: >>>>>> http://news.gmane.org/gmane.**science.biology.informatics.**con ductor<http: news.gmane.org="" gmane.science.biology.informatics.conduct="" or=""> >>>>>> >>>>>> [[alternative HTML version deleted]] >>>>> >>>>> ______________________________**_________________ >>>>> Bioconductor mailing list >>>>> Bioconductor@r-project.org >>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: sta="" t.ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>>> Search the archives: >>>>> http://news.gmane.org/gmane.**science.biology.informatics.**cond uctor<http: news.gmane.org="" gmane.science.biology.informatics.conducto="" r=""> >>>>> >>>>> >>>> >>>> -- >>>> Dr. Suparna Mitra >>>> Wolfson Centre for Personalised Medicine >>>> Department of Molecular and Clinical Pharmacology >>>> Institute of Translational Medicine University of Liverpool >>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >>>> >>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >>>> M: +44 (0) 7511387895 >>>> Email id: smitra@liverpool.ac.uk >>>> Alternative Email id: suparna.mitra.sm@gmail.com >>>> >>>> ______________________________**_________________ >>>> Bioconductor mailing list >>>> Bioconductor@r-project.org >>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat="" .ethz.ch="" mailman="" listinfo="" bioconductor=""> >>>> Search the archives: >>>> http://news.gmane.org/gmane.**science.biology.informatics.**condu ctor<http: news.gmane.org="" gmane.science.biology.informatics.conductor=""> >>>> >>>> [[alternative HTML version deleted]] >>> >>> ______________________________**_________________ >>> Bioconductor mailing list >>> Bioconductor@r-project.org >>> https://stat.ethz.ch/mailman/**listinfo/bioconductor<https: stat.="" ethz.ch="" mailman="" listinfo="" bioconductor=""> >>> Search the archives: http://news.gmane.org/gmane.** >>> science.biology.informatics.**conductor<http: news.gmane.org="" gman="" e.science.biology.informatics.conductor=""> >>> >> >> -- >> James W. MacDonald, M.S. >> Biostatistician >> University of Washington >> Environmental and Occupational Health Sciences >> 4225 Roosevelt Way NE, # 100 >> Seattle WA 98105-6099 >> >> > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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On Mon, Sep 10, 2012 at 1:57 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > Hello group, > > Can anybody please you please suggest how I can do the following in BioC to > match a previous set of experiment. > #Entitylist : A v R Filtered on Error - CV < 300.0 percent > #Interpretation : A vs R Treatment > #Experiment: RMA > #p-value cut-off:0.05 > #Selected Test : T Test unpaired > #p-value computation: Asymptotic > > Until now I have done rma on direct data and performed t-test. But how can > I 'Filtered on Error - CV' and select p-value computation as 'Asymptotic'? > > This is what I did. > > > sessionInfo() > R version 2.15.1 (2012-06-22) > Platform: i386-apple-darwin9.8.0/i386 (32-bit) > > locale: > [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] statmod_1.4.15 limma_3.12.1 > annotate_1.34.1 hugene10stprobeset.db_8.0.1 org.Hs.eg.db_2.7.1 > > [6] BiocInstaller_1.4.7 affycoretools_1.28.0 KEGG.db_2.7.1 > GO.db_2.7.1 AnnotationDbi_1.18.1 > [11] affy_1.34.0 Biobase_2.16.0 > BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 > > [16] DBI_0.2-5 oligo_1.20.4 > oligoClasses_1.18.0 > > > rmaOligoinvivo = oligo::rma(InVivodat1) > Background correcting > Normalizing > Calculating Expression > > > rmaOligoinvitro = oligo::rma(InVitrodat1) > Background correcting > Normalizing > Calculating Expression > > > maplot(rmaOligoinvivo) > > maplot(rmaOligoinvitro) > > InVivoTargets > FileName Treatment > 1 MC1 A > 2 MC2 A > 3 MC3 A > 4 MC4 A > 5 MC5 A > 6 MC6 A > 7 MC7 R > 8 MC8 R > 9 MC9 R > 10 MC10 R > 11 MC11 R > 12 MC12 R > 13 MC13 T > 14 MC14 T > 15 MC15 T > 16 MC16 T > 17 MC17 T > 18 MC18 T > > InVitroTargets > FileName Treatment Batch CD4 > 1 MC19 RY 1 High > 2 MC20 TY 1 Low > 3 MC21 RY 2 High > 4 MC22 TY 2 High > 5 MC23 TY 2 Low > 6 MC24 RY 2 High > 7 MC25 TXY 1 Low > 8 MC26 RXY 1 High > 9 MC27 RXY 2 Low > 10 MC28 TXY 2 High11 MC29 RXY 2 High > 12 MC30 TXY 2 High > > f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) > > design.invivo <- model.matrix(~0 + f.invivo) > > > > > > colnames(design.invivo) <- c("A", "R", "T") > > Just subset the rmaOligoinvivo object by CV. rmaOligoinvivo = rmaOligoinvivo[apply(exprs(rmaOligoinvivo)),1,function(x) {sd(x)/mean(x)})>3,] This is untested but, hopefully, you can get the idea and can implement a solution that works for you. I'm not sure where the cutoff of 3 (300%) comes from, so you may find that you need to adjust that to your data. Sean > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > > > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = > design.invivo) > > > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) > > > fit2.invivo <-eBayes(fit2.invivo) > > > topTable(fit2.invivo, coef = 1, adjust = "fdr") > > ID logFC AveExpr t P.Value adj.P.Val > B > > 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > > 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > -2.023533 > ... > > Thanks a lot, > Mitra > > > > > > On 7 September 2012 14:55, James W. MacDonald <jmacdon@uw.edu> wrote: > > > >> > >> > >> On 9/7/2012 7:26 AM, Sean Davis wrote: > >> > >>> On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra<smitra@liverpool.ac.uk> >** > >>> wrote: > >>> > >>> Oh thanks.. I missed this point. But can you suggest me one more > >>>> thing... > >>>> when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the > same > >>>> result as adjust = "fdr". for topTable. Is it normal? > >>>> > >>>> Yes. They are the same. See the help for p.adjust for details. > >>> > >>> > >>> Further when I tried to do vennDiagram I was surprized to see 0 in all > >>>> circles. Thus I thought I must be doing something wrong. Sorry if my > >>>> question is silly. > >>>> > >>>> Unfortunately, you have no significantly differentially- expressed > >>> genes. > >>> Note that all of the adjusted p-values are pretty high. You can try > to > >>> filter your genes based on variance before testing to try to reduce the > >>> number of genes entering your test and multiple correction. However, > >>> having worked with this kind of biological system (patients), you may > >>> suffering from a problem of a small biological effect in the setting of > >>> large biological variation. A larger sample size may be necessary. > >>> > >> > >> You may also be suffering from large technical variation, which could be > >> helped by applying array weights. See ?arrayWeights for more > information. > >> > >> Best, > >> > >> Jim > >> > >> > >> > >>> Sean > >>> > >>> > >>> Here is what I tried. > >>>> > >>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") > >>>>> > >>>> ID logFC AveExpr t P.Value adj.P.Val > >>>> B > >>>> > >>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >>>> -2.023533 > >>>> > >>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >>>> -2.023533 > >>>> > >>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >>>> -2.023533 > >>>> > >>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >>>> -2.023533 > >>>> > >>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > >>>> -2.323922 > >>>> > >>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > >>>> -2.454618 > >>>> > >>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > >>>> -2.607991 > >>>> > >>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > >>>> -2.685960 > >>>> > >>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > >>>> -2.711203 > >>>> > >>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > >>>> -2.800385 > >>>> > >>>> topTable(fit2.invivo, coef = 2, adjust = "fdr") > >>>>> > >>>> ID logFC AveExpr t P.Value adj.P.Val > >>>> B > >>>> > >>>> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 > >>>> -2.469821 > >>>> > >>>> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 > >>>> -2.652426 > >>>> > >>>> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 > >>>> -2.662042 > >>>> > >>>> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 > >>>> -2.702782 > >>>> > >>>> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 > >>>> -2.707492 > >>>> > >>>> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 > >>>> -2.717698 > >>>> > >>>> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 > >>>> -2.734873 > >>>> > >>>> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 > >>>> -2.772143 > >>>> > >>>> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 > >>>> -2.779941 > >>>> > >>>> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 > >>>> -2.785214 > >>>> > >>>> topTable(fit2.invivo, coef = 3, adjust = "fdr") > >>>>> > >>>> ID logFC AveExpr t P.Value adj.P.Val > >>>> B > >>>> > >>>> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 > >>>> -0.6982314 > >>>> > >>>> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 > >>>> -1.2472882 > >>>> > >>>> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 > >>>> -1.2727646 > >>>> > >>>> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 > >>>> -1.2831553 > >>>> > >>>> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 > >>>> -1.4031177 > >>>> > >>>> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 > >>>> -1.4733549 > >>>> > >>>> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 > >>>> -1.5025187 > >>>> > >>>> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 > >>>> -1.5612836 > >>>> > >>>> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 > >>>> -1.6535466 > >>>> > >>>> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 > >>>> -1.6662493 > >>>> > >>>> results<- decideTests(fit2.invivo) > >>>>> vennDiagram(results) > >>>>> > >>>> see the plot attached. > >>>> Thanks, > >>>> Mitra > >>>> > >>>> > >>>> On 7 September 2012 12:03, Sean Davis<sdavis2@mail.nih.gov> wrote: > >>>> > >>>> On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra<smitra@liverpool.ac.uk> >>>>> > >>>>>> wrote: > >>>>>> Dear Sean, > >>>>>> I have been reading Bioconductor and limma user guide and thus > this > >>>>>> > >>>>> is > >>>> > >>>>> I > >>>>> > >>>>>> tried. > >>>>>> But just being a novice, wanted to make sure if I am right. > >>>>>> I know I have perform t-test when I created the contrast, but can > you > >>>>>> please help me how can I perform unpaired t-test here. I am > concerned > >>>>>> > >>>>> as > >>>> > >>>>> the patients in groups are not same. > >>>>>> > >>>>>> > >>>>> The t-test you performed was unpaired; unpaired is the "default". > >>>>> > >>>>> Sean > >>>>> > >>>>> > >>>>> Thanks, > >>>>>> Mitra > >>>>>> > >>>>>> On 7 September 2012 11:41, Sean Davis<sdavis2@mail.nih.gov> wrote: > >>>>>> > >>>>>> > >>>>>>> On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra< > >>>>>>> > >>>>>> smitra@liverpool.ac.uk > >>>> > >>>>> wrote: > >>>>>>> > >>>>>>> Hello Group, > >>>>>>>> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using > >>>>>>>> > >>>>>>> BiC. > >>>> > >>>>> Previously i was using different softwares for this. And this is my > >>>>>>>> > >>>>>>> first > >>>>>> > >>>>>>> try with Bioconductor for big experiment. So thought to get some > >>>>>>>> > >>>>>>> advice > >>>>> > >>>>>> in > >>>>>> > >>>>>>> the beginning. > >>>>>>>> I have Three groups of patient: (In-vivo) > >>>>>>>> A-Acute reaction. Patient taking a drug X develops reaction. > >>>>>>>> R-recovered (6 weeks after acute reaction-not longer taking drug > >>>>>>>> > >>>>>>> X). > >>>> > >>>>> T-Tolerant. Patient on X and tolerating treatment. > >>>>>>>> > >>>>>>>> Now in in-vitro study we used another constant Y > >>>>>>>> RXY recovered and challenged with X+Y > >>>>>>>> RY recovered challenged with only Y. RXY vs RY are to exclude > >>>>>>>> > >>>>>>> effects > >>>>> > >>>>>> by > >>>>>>>> Y. > >>>>>>>> TXY tolerant and challenged with X+Y, > >>>>>>>> TY tolerant challenged with only Y. TXY vs TY are to exclude > >>>>>>>> > >>>>>>> effects > >>>> > >>>>> by > >>>>>> > >>>>>>> Y. > >>>>>>>> > >>>>>>>> No I want to check the cross relation and effects A vs R, RvsT and > >>>>>>>> > >>>>>>> Avs T > >>>>> > >>>>>> and differentially expressed genes for each comparison. And the > >>>>>>>> > >>>>>>> same > >>>> > >>>>> in > >>>>> > >>>>>> invitro. There are not same patients in different groups, thus I > >>>>>>>> > >>>>>>> think I > >>>>> > >>>>>> want to apply unpaired-t test. > >>>>>>>> > >>>>>>>> This is what I tried: > >>>>>>>> > >>>>>>>>> sessionInfo() > >>>>>>>>> > >>>>>>>> R version 2.15.1 (2012-06-22) > >>>>>>>> Platform: i386-apple-darwin9.8.0/i386 (32-bit) > >>>>>>>> > >>>>>>>> locale: > >>>>>>>> [1] > en_GB.UTF-8/en_GB.UTF-8/en_GB.**UTF-8/C/en_GB.UTF-8/en_GB.UTF-* > >>>>>>>> *8 > >>>>>>>> > >>>>>>>> attached base packages: > >>>>>>>> [1] stats graphics grDevices utils datasets methods > base > >>>>>>>> > >>>>>>>> other attached packages: > >>>>>>>> [1] statmod_1.4.15 limma_3.12.1 > >>>>>>>> annotate_1.34.1 hugene10stprobeset.db_8.0.1 > >>>>>>>> org.Hs.eg.db_2.7.1 > >>>>>>>> > >>>>>>>> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 > >>>>>>>> > >>>>>>> KEGG.db_2.7.1 > >>>>>> > >>>>>>> GO.db_2.7.1 AnnotationDbi_1.18.1 > >>>>>>>> [11] affy_1.34.0 Biobase_2.16.0 > >>>>>>>> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 > >>>>>>>> > >>>>>>> RSQLite_0.11.1 > >>>>> > >>>>>> [16] DBI_0.2-5 oligo_1.20.4 > >>>>>>>> oligoClasses_1.18.0 > >>>>>>>> > >>>>>>>> > >>>>>>>> rmaOligoinvivo = oligo::rma(InVivodat1) > >>>>>>>> Background correcting > >>>>>>>> Normalizing > >>>>>>>> Calculating Expression > >>>>>>>> > >>>>>>>> rmaOligoinvitro = oligo::rma(InVitrodat1) > >>>>>>>>> > >>>>>>>> Background correcting > >>>>>>>> Normalizing > >>>>>>>> Calculating Expression > >>>>>>>> > >>>>>>>> maplot(rmaOligoinvivo) > >>>>>>>>> maplot(rmaOligoinvitro) > >>>>>>>>> InVivoTargets > >>>>>>>>> > >>>>>>>> FileName Treatment > >>>>>>>> 1 MC1 A > >>>>>>>> 2 MC2 A > >>>>>>>> 3 MC3 A > >>>>>>>> 4 MC4 A > >>>>>>>> 5 MC5 A > >>>>>>>> 6 MC6 A > >>>>>>>> 7 MC7 R > >>>>>>>> 8 MC8 R > >>>>>>>> 9 MC9 R > >>>>>>>> 10 MC10 R > >>>>>>>> 11 MC11 R > >>>>>>>> 12 MC12 R > >>>>>>>> 13 MC13 T > >>>>>>>> 14 MC14 T > >>>>>>>> 15 MC15 T > >>>>>>>> 16 MC16 T > >>>>>>>> 17 MC17 T > >>>>>>>> 18 MC18 T > >>>>>>>> > >>>>>>>> InVitroTargets=readTargets("~/**Desktop/Recent/Liverpool- work-** > >>>> related/Micro_RawData/**InVitroTargets.txt") > >>>> > >>>>> InVitroTargets > >>>>>>>>> > >>>>>>>> FileName Treatment Batch CD4 > >>>>>>>> 1 MC19 RY 1 High > >>>>>>>> 2 MC20 TY 1 Low > >>>>>>>> 3 MC21 RY 2 High > >>>>>>>> 4 MC22 TY 2 High > >>>>>>>> 5 MC23 TY 2 Low > >>>>>>>> 6 MC24 RY 2 High > >>>>>>>> 7 MC25 TXY 1 Low > >>>>>>>> 8 MC26 RXY 1 High > >>>>>>>> 9 MC27 RXY 2 Low > >>>>>>>> 10 MC28 TXY 2 High > >>>>>>>> 11 MC29 RXY 2 High > >>>>>>>> 12 MC30 TXY 2 High > >>>>>>>> > >>>>>>>> f.invivo<- factor(InVivoTargets$**Treatment, levels = c("A", "R", > >>>>>>>> > >>>>>>> "T")) > >>>> > >>>>> design.invivo<- model.matrix(~0 + f.invivo) > >>>>>>>> > >>>>>>>> colnames(design.invivo)<- c("A", "R", "T") > >>>>>>>>> fit.invivo<- lmFit(rmaOligoinvivo, design.invivo) > >>>>>>>>> contrast.matrix.invivo<- makeContrasts(R-A, T-R, T-A,levels = > >>>>>>>>> > >>>>>>>> design.invivo) > >>>>>>>> > >>>>>>>> fit2.invivo<- contrasts.fit(fit.invivo, contrast.matrix.invivo) > >>>>>>>>> fit2.invivo<-eBayes(fit2.**invivo) > >>>>>>>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") > >>>>>>>>> > >>>>>>>> ID logFC AveExpr t P.Value > adj.P.Val > >>>>>>>> B > >>>>>>>> > >>>>>>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >>>>>>>> -2.023533 > >>>>>>>> > >>>>>>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >>>>>>>> -2.023533 > >>>>>>>> > >>>>>>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >>>>>>>> -2.023533 > >>>>>>>> > >>>>>>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >>>>>>>> -2.023533 > >>>>>>>> > >>>>>>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 > 0.9552974 > >>>>>>>> -2.323922 > >>>>>>>> > >>>>>>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 > 0.9998796 > >>>>>>>> -2.454618 > >>>>>>>> > >>>>>>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 > 0.9998796 > >>>>>>>> -2.607991 > >>>>>>>> > >>>>>>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 > 0.9998796 > >>>>>>>> -2.685960 > >>>>>>>> > >>>>>>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 > 0.9998796 > >>>>>>>> -2.711203 > >>>>>>>> > >>>>>>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 > 0.9998796 > >>>>>>>> -2.800385 > >>>>>>>> I am progressing in a right way? Further I want to perform > unpaired > >>>>>>>> > >>>>>>> t > >>>> > >>>>> test > >>>>>> > >>>>>>> for comparing AvsT and so on. Any help will be really great. > >>>>>>>> > >>>>>>>> Hi, Mitra. I think that looks about right. You have already > >>>>>>> > >>>>>> performed > >>>> > >>>>> the unpaired t-test of AvsT (well, actually TvsA, but the p-values > >>>>>>> > >>>>>> will > >>>> > >>>>> be > >>>>>> > >>>>>>> the same) as coefficient 3. > >>>>>>> > >>>>>>> Sean > >>>>>>> > >>>>>>> > >>>>>>> > >>>>>> > >>>>>> -- > >>>>>> Dr. Suparna Mitra > >>>>>> Wolfson Centre for Personalised Medicine > >>>>>> Department of Molecular and Clinical Pharmacology > >>>>>> Institute of Translational Medicine University of Liverpool > >>>>>> Block A: Waterhouse Buildings, L69 3GL Liverpool > >>>>>> > >>>>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 > >>>>>> M: +44 (0) 7511387895 > >>>>>> Email id: smitra@liverpool.ac.uk > >>>>>> Alternative Email id: suparna.mitra.sm@gmail.com > >>>>>> > >>>>>> [[alternative HTML version deleted]] > >>>>>> > >>>>>> ______________________________**_________________ > >>>>>> Bioconductor mailing list > >>>>>> Bioconductor@r-project.org > >>>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< > https://stat.ethz.ch/mailman/listinfo/bioconductor> > >>>>>> Search the archives: > >>>>>> > http://news.gmane.org/gmane.**science.biology.informatics.**conductor< > http://news.gmane.org/gmane.science.biology.informatics.conductor> > >>>>>> > >>>>>> [[alternative HTML version deleted]] > >>>>> > >>>>> ______________________________**_________________ > >>>>> Bioconductor mailing list > >>>>> Bioconductor@r-project.org > >>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< > https://stat.ethz.ch/mailman/listinfo/bioconductor> > >>>>> Search the archives: > >>>>> > http://news.gmane.org/gmane.**science.biology.informatics.**conductor< > http://news.gmane.org/gmane.science.biology.informatics.conductor> > >>>>> > >>>>> > >>>> > >>>> -- > >>>> Dr. Suparna Mitra > >>>> Wolfson Centre for Personalised Medicine > >>>> Department of Molecular and Clinical Pharmacology > >>>> Institute of Translational Medicine University of Liverpool > >>>> Block A: Waterhouse Buildings, L69 3GL Liverpool > >>>> > >>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 > >>>> M: +44 (0) 7511387895 > >>>> Email id: smitra@liverpool.ac.uk > >>>> Alternative Email id: suparna.mitra.sm@gmail.com > >>>> > >>>> ______________________________**_________________ > >>>> Bioconductor mailing list > >>>> Bioconductor@r-project.org > >>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< > https://stat.ethz.ch/mailman/listinfo/bioconductor> > >>>> Search the archives: > >>>> http://news.gmane.org/gmane.**science.biology.informatics.**conductor > <http: news.gmane.org="" gmane.science.biology.informatics.conductor=""> > >>>> > >>>> [[alternative HTML version deleted]] > >>> > >>> ______________________________**_________________ > >>> Bioconductor mailing list > >>> Bioconductor@r-project.org > >>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< > https://stat.ethz.ch/mailman/listinfo/bioconductor> > >>> Search the archives: http://news.gmane.org/gmane.** > >>> science.biology.informatics.**conductor< > http://news.gmane.org/gmane.science.biology.informatics.conductor> > >>> > >> > >> -- > >> James W. MacDonald, M.S. > >> Biostatistician > >> University of Washington > >> Environmental and Occupational Health Sciences > >> 4225 Roosevelt Way NE, # 100 > >> Seattle WA 98105-6099 > >> > >> > > > > > > -- > > Dr. Suparna Mitra > > Wolfson Centre for Personalised Medicine > > Department of Molecular and Clinical Pharmacology > > Institute of Translational Medicine University of Liverpool > > Block A: Waterhouse Buildings, L69 3GL Liverpool > > > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > > M: +44 (0) 7511387895 > > Email id: smitra@liverpool.ac.uk > > Alternative Email id: suparna.mitra.sm@gmail.com > > > > > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor@r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]
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Thanks a lot Sean. I will try this. Best wishes, Suparna. On 10 September 2012 11:57, Sean Davis <sdavis2@mail.nih.gov> wrote: > > > On Mon, Sep 10, 2012 at 1:57 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > >> Hello group, >> >> Can anybody please you please suggest how I can do the following in BioC >> to >> match a previous set of experiment. >> #Entitylist : A v R Filtered on Error - CV < 300.0 percent >> #Interpretation : A vs R Treatment >> #Experiment: RMA >> #p-value cut-off:0.05 >> #Selected Test : T Test unpaired >> #p-value computation: Asymptotic >> >> Until now I have done rma on direct data and performed t-test. But how can >> I 'Filtered on Error - CV' and select p-value computation as 'Asymptotic'? >> >> This is what I did. >> >> > sessionInfo() >> R version 2.15.1 (2012-06-22) >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >> >> locale: >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 >> >> attached base packages: >> [1] stats graphics grDevices utils datasets methods base >> >> other attached packages: >> [1] statmod_1.4.15 limma_3.12.1 >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >> org.Hs.eg.db_2.7.1 >> >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 KEGG.db_2.7.1 >> GO.db_2.7.1 AnnotationDbi_1.18.1 >> [11] affy_1.34.0 Biobase_2.16.0 >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 RSQLite_0.11.1 >> >> [16] DBI_0.2-5 oligo_1.20.4 >> oligoClasses_1.18.0 >> >> >> rmaOligoinvivo = oligo::rma(InVivodat1) >> Background correcting >> Normalizing >> Calculating Expression >> >> > rmaOligoinvitro = oligo::rma(InVitrodat1) >> Background correcting >> Normalizing >> Calculating Expression >> >> > maplot(rmaOligoinvivo) >> > maplot(rmaOligoinvitro) >> > InVivoTargets >> FileName Treatment >> 1 MC1 A >> 2 MC2 A >> 3 MC3 A >> 4 MC4 A >> 5 MC5 A >> 6 MC6 A >> 7 MC7 R >> 8 MC8 R >> 9 MC9 R >> 10 MC10 R >> 11 MC11 R >> 12 MC12 R >> 13 MC13 T >> 14 MC14 T >> 15 MC15 T >> 16 MC16 T >> 17 MC17 T >> 18 MC18 T >> > InVitroTargets >> FileName Treatment Batch CD4 >> 1 MC19 RY 1 High >> 2 MC20 TY 1 Low >> 3 MC21 RY 2 High >> 4 MC22 TY 2 High >> 5 MC23 TY 2 Low >> 6 MC24 RY 2 High >> 7 MC25 TXY 1 Low >> 8 MC26 RXY 1 High >> 9 MC27 RXY 2 Low >> 10 MC28 TXY 2 High11 MC29 RXY 2 High >> 12 MC30 TXY 2 High >> >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", "T")) >> >> design.invivo <- model.matrix(~0 + f.invivo) >> >> > >> >> > colnames(design.invivo) <- c("A", "R", "T") >> >> > Just subset the rmaOligoinvivo object by CV. > > rmaOligoinvivo = rmaOligoinvivo[apply(exprs(rmaOligoinvivo)),1,function(x) > {sd(x)/mean(x)})>3,] > > This is untested but, hopefully, you can get the idea and can implement a > solution that works for you. I'm not sure where the cutoff of 3 (300%) > comes from, so you may find that you need to adjust that to your data. > > Sean > > > > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) >> >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = >> design.invivo) >> >> > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) >> >> > fit2.invivo <-eBayes(fit2.invivo) >> >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") >> >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> ... >> >> Thanks a lot, >> Mitra >> >> >> > >> > On 7 September 2012 14:55, James W. MacDonald <jmacdon@uw.edu> wrote: >> > >> >> >> >> >> >> On 9/7/2012 7:26 AM, Sean Davis wrote: >> >> >> >>> On Fri, Sep 7, 2012 at 7:14 AM, suparna mitra<smitra@liverpool.ac.uk>> >** >> >> >>> wrote: >> >>> >> >>> Oh thanks.. I missed this point. But can you suggest me one more >> >>>> thing... >> >>>> when I tried adjust = "BH" (Benjamini-Hochberg I suppose) I got the >> same >> >>>> result as adjust = "fdr". for topTable. Is it normal? >> >>>> >> >>>> Yes. They are the same. See the help for p.adjust for details. >> >>> >> >>> >> >>> Further when I tried to do vennDiagram I was surprized to see 0 in >> all >> >>>> circles. Thus I thought I must be doing something wrong. Sorry if my >> >>>> question is silly. >> >>>> >> >>>> Unfortunately, you have no significantly differentially- expressed >> >>> genes. >> >>> Note that all of the adjusted p-values are pretty high. You can >> try to >> >>> filter your genes based on variance before testing to try to reduce >> the >> >>> number of genes entering your test and multiple correction. However, >> >>> having worked with this kind of biological system (patients), you may >> >>> suffering from a problem of a small biological effect in the setting >> of >> >>> large biological variation. A larger sample size may be necessary. >> >>> >> >> >> >> You may also be suffering from large technical variation, which could >> be >> >> helped by applying array weights. See ?arrayWeights for more >> information. >> >> >> >> Best, >> >> >> >> Jim >> >> >> >> >> >> >> >>> Sean >> >>> >> >>> >> >>> Here is what I tried. >> >>>> >> >>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >> >>>>> >> >>>> ID logFC AveExpr t P.Value adj.P.Val >> >>>> B >> >>>> >> >>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> >>>> -2.023533 >> >>>> >> >>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> >>>> -2.023533 >> >>>> >> >>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> >>>> -2.023533 >> >>>> >> >>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> >>>> -2.023533 >> >>>> >> >>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >> >>>> -2.323922 >> >>>> >> >>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >> >>>> -2.454618 >> >>>> >> >>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >> >>>> -2.607991 >> >>>> >> >>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >> >>>> -2.685960 >> >>>> >> >>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >> >>>> -2.711203 >> >>>> >> >>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >> >>>> -2.800385 >> >>>> >> >>>> topTable(fit2.invivo, coef = 2, adjust = "fdr") >> >>>>> >> >>>> ID logFC AveExpr t P.Value adj.P.Val >> >>>> B >> >>>> >> >>>> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 >> >>>> -2.469821 >> >>>> >> >>>> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 >> >>>> -2.652426 >> >>>> >> >>>> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 >> >>>> -2.662042 >> >>>> >> >>>> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 >> >>>> -2.702782 >> >>>> >> >>>> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 >> >>>> -2.707492 >> >>>> >> >>>> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 >> >>>> -2.717698 >> >>>> >> >>>> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 >> >>>> -2.734873 >> >>>> >> >>>> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 >> >>>> -2.772143 >> >>>> >> >>>> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 >> >>>> -2.779941 >> >>>> >> >>>> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 >> >>>> -2.785214 >> >>>> >> >>>> topTable(fit2.invivo, coef = 3, adjust = "fdr") >> >>>>> >> >>>> ID logFC AveExpr t P.Value adj.P.Val >> >>>> B >> >>>> >> >>>> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 >> >>>> -0.6982314 >> >>>> >> >>>> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 >> >>>> -1.2472882 >> >>>> >> >>>> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 >> >>>> -1.2727646 >> >>>> >> >>>> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 >> >>>> -1.2831553 >> >>>> >> >>>> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 >> >>>> -1.4031177 >> >>>> >> >>>> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 >> >>>> -1.4733549 >> >>>> >> >>>> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 >> >>>> -1.5025187 >> >>>> >> >>>> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 >> >>>> -1.5612836 >> >>>> >> >>>> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 >> >>>> -1.6535466 >> >>>> >> >>>> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 >> >>>> -1.6662493 >> >>>> >> >>>> results<- decideTests(fit2.invivo) >> >>>>> vennDiagram(results) >> >>>>> >> >>>> see the plot attached. >> >>>> Thanks, >> >>>> Mitra >> >>>> >> >>>> >> >>>> On 7 September 2012 12:03, Sean Davis<sdavis2@mail.nih.gov> wrote: >> >>>> >> >>>> On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra< >> smitra@liverpool.ac.uk >> >>>>> >> >>>>>> wrote: >> >>>>>> Dear Sean, >> >>>>>> I have been reading Bioconductor and limma user guide and thus >> this >> >>>>>> >> >>>>> is >> >>>> >> >>>>> I >> >>>>> >> >>>>>> tried. >> >>>>>> But just being a novice, wanted to make sure if I am right. >> >>>>>> I know I have perform t-test when I created the contrast, but can >> you >> >>>>>> please help me how can I perform unpaired t-test here. I am >> concerned >> >>>>>> >> >>>>> as >> >>>> >> >>>>> the patients in groups are not same. >> >>>>>> >> >>>>>> >> >>>>> The t-test you performed was unpaired; unpaired is the "default". >> >>>>> >> >>>>> Sean >> >>>>> >> >>>>> >> >>>>> Thanks, >> >>>>>> Mitra >> >>>>>> >> >>>>>> On 7 September 2012 11:41, Sean Davis<sdavis2@mail.nih.gov> >> wrote: >> >>>>>> >> >>>>>> >> >>>>>>> On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra< >> >>>>>>> >> >>>>>> smitra@liverpool.ac.uk >> >>>> >> >>>>> wrote: >> >>>>>>> >> >>>>>>> Hello Group, >> >>>>>>>> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using >> >>>>>>>> >> >>>>>>> BiC. >> >>>> >> >>>>> Previously i was using different softwares for this. And this is my >> >>>>>>>> >> >>>>>>> first >> >>>>>> >> >>>>>>> try with Bioconductor for big experiment. So thought to get some >> >>>>>>>> >> >>>>>>> advice >> >>>>> >> >>>>>> in >> >>>>>> >> >>>>>>> the beginning. >> >>>>>>>> I have Three groups of patient: (In-vivo) >> >>>>>>>> A-Acute reaction. Patient taking a drug X develops reaction. >> >>>>>>>> R-recovered (6 weeks after acute reaction-not longer taking >> drug >> >>>>>>>> >> >>>>>>> X). >> >>>> >> >>>>> T-Tolerant. Patient on X and tolerating treatment. >> >>>>>>>> >> >>>>>>>> Now in in-vitro study we used another constant Y >> >>>>>>>> RXY recovered and challenged with X+Y >> >>>>>>>> RY recovered challenged with only Y. RXY vs RY are to exclude >> >>>>>>>> >> >>>>>>> effects >> >>>>> >> >>>>>> by >> >>>>>>>> Y. >> >>>>>>>> TXY tolerant and challenged with X+Y, >> >>>>>>>> TY tolerant challenged with only Y. TXY vs TY are to exclude >> >>>>>>>> >> >>>>>>> effects >> >>>> >> >>>>> by >> >>>>>> >> >>>>>>> Y. >> >>>>>>>> >> >>>>>>>> No I want to check the cross relation and effects A vs R, RvsT >> and >> >>>>>>>> >> >>>>>>> Avs T >> >>>>> >> >>>>>> and differentially expressed genes for each comparison. And the >> >>>>>>>> >> >>>>>>> same >> >>>> >> >>>>> in >> >>>>> >> >>>>>> invitro. There are not same patients in different groups, thus I >> >>>>>>>> >> >>>>>>> think I >> >>>>> >> >>>>>> want to apply unpaired-t test. >> >>>>>>>> >> >>>>>>>> This is what I tried: >> >>>>>>>> >> >>>>>>>>> sessionInfo() >> >>>>>>>>> >> >>>>>>>> R version 2.15.1 (2012-06-22) >> >>>>>>>> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >> >>>>>>>> >> >>>>>>>> locale: >> >>>>>>>> [1] >> en_GB.UTF-8/en_GB.UTF-8/en_GB.**UTF-8/C/en_GB.UTF-8/en_GB.UTF-* >> >> >>>>>>>> *8 >> >>>>>>>> >> >>>>>>>> attached base packages: >> >>>>>>>> [1] stats graphics grDevices utils datasets methods >> base >> >>>>>>>> >> >>>>>>>> other attached packages: >> >>>>>>>> [1] statmod_1.4.15 limma_3.12.1 >> >>>>>>>> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >> >>>>>>>> org.Hs.eg.db_2.7.1 >> >>>>>>>> >> >>>>>>>> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 >> >>>>>>>> >> >>>>>>> KEGG.db_2.7.1 >> >>>>>> >> >>>>>>> GO.db_2.7.1 AnnotationDbi_1.18.1 >> >>>>>>>> [11] affy_1.34.0 Biobase_2.16.0 >> >>>>>>>> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 >> >>>>>>>> >> >>>>>>> RSQLite_0.11.1 >> >>>>> >> >>>>>> [16] DBI_0.2-5 oligo_1.20.4 >> >>>>>>>> oligoClasses_1.18.0 >> >>>>>>>> >> >>>>>>>> >> >>>>>>>> rmaOligoinvivo = oligo::rma(InVivodat1) >> >>>>>>>> Background correcting >> >>>>>>>> Normalizing >> >>>>>>>> Calculating Expression >> >>>>>>>> >> >>>>>>>> rmaOligoinvitro = oligo::rma(InVitrodat1) >> >>>>>>>>> >> >>>>>>>> Background correcting >> >>>>>>>> Normalizing >> >>>>>>>> Calculating Expression >> >>>>>>>> >> >>>>>>>> maplot(rmaOligoinvivo) >> >>>>>>>>> maplot(rmaOligoinvitro) >> >>>>>>>>> InVivoTargets >> >>>>>>>>> >> >>>>>>>> FileName Treatment >> >>>>>>>> 1 MC1 A >> >>>>>>>> 2 MC2 A >> >>>>>>>> 3 MC3 A >> >>>>>>>> 4 MC4 A >> >>>>>>>> 5 MC5 A >> >>>>>>>> 6 MC6 A >> >>>>>>>> 7 MC7 R >> >>>>>>>> 8 MC8 R >> >>>>>>>> 9 MC9 R >> >>>>>>>> 10 MC10 R >> >>>>>>>> 11 MC11 R >> >>>>>>>> 12 MC12 R >> >>>>>>>> 13 MC13 T >> >>>>>>>> 14 MC14 T >> >>>>>>>> 15 MC15 T >> >>>>>>>> 16 MC16 T >> >>>>>>>> 17 MC17 T >> >>>>>>>> 18 MC18 T >> >>>>>>>> >> >>>>>>>> InVitroTargets=readTargets("~/**Desktop/Recent/Liverpool- work-** >> >>>> related/Micro_RawData/**InVitroTargets.txt") >> >> >>>> >> >>>>> InVitroTargets >> >>>>>>>>> >> >>>>>>>> FileName Treatment Batch CD4 >> >>>>>>>> 1 MC19 RY 1 High >> >>>>>>>> 2 MC20 TY 1 Low >> >>>>>>>> 3 MC21 RY 2 High >> >>>>>>>> 4 MC22 TY 2 High >> >>>>>>>> 5 MC23 TY 2 Low >> >>>>>>>> 6 MC24 RY 2 High >> >>>>>>>> 7 MC25 TXY 1 Low >> >>>>>>>> 8 MC26 RXY 1 High >> >>>>>>>> 9 MC27 RXY 2 Low >> >>>>>>>> 10 MC28 TXY 2 High >> >>>>>>>> 11 MC29 RXY 2 High >> >>>>>>>> 12 MC30 TXY 2 High >> >>>>>>>> >> >>>>>>>> f.invivo<- factor(InVivoTargets$**Treatment, levels = c("A", "R", >> >> >>>>>>>> >> >>>>>>> "T")) >> >>>> >> >>>>> design.invivo<- model.matrix(~0 + f.invivo) >> >>>>>>>> >> >>>>>>>> colnames(design.invivo)<- c("A", "R", "T") >> >>>>>>>>> fit.invivo<- lmFit(rmaOligoinvivo, design.invivo) >> >>>>>>>>> contrast.matrix.invivo<- makeContrasts(R-A, T-R, T-A,levels = >> >>>>>>>>> >> >>>>>>>> design.invivo) >> >>>>>>>> >> >>>>>>>> fit2.invivo<- contrasts.fit(fit.invivo, contrast.matrix.invivo) >> >>>>>>>>> fit2.invivo<-eBayes(fit2.**invivo) >> >> >>>>>>>>> topTable(fit2.invivo, coef = 1, adjust = "fdr") >> >>>>>>>>> >> >>>>>>>> ID logFC AveExpr t P.Value >> adj.P.Val >> >>>>>>>> B >> >>>>>>>> >> >>>>>>>> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 >> 0.3282013 >> >>>>>>>> -2.023533 >> >>>>>>>> >> >>>>>>>> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 >> 0.3282013 >> >>>>>>>> -2.023533 >> >>>>>>>> >> >>>>>>>> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 >> 0.3282013 >> >>>>>>>> -2.023533 >> >>>>>>>> >> >>>>>>>> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 >> 0.3282013 >> >>>>>>>> -2.023533 >> >>>>>>>> >> >>>>>>>> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 >> 0.9552974 >> >>>>>>>> -2.323922 >> >>>>>>>> >> >>>>>>>> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 >> 0.9998796 >> >>>>>>>> -2.454618 >> >>>>>>>> >> >>>>>>>> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 >> 0.9998796 >> >>>>>>>> -2.607991 >> >>>>>>>> >> >>>>>>>> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 >> 0.9998796 >> >>>>>>>> -2.685960 >> >>>>>>>> >> >>>>>>>> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 >> 0.9998796 >> >>>>>>>> -2.711203 >> >>>>>>>> >> >>>>>>>> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 >> 0.9998796 >> >>>>>>>> -2.800385 >> >>>>>>>> I am progressing in a right way? Further I want to perform >> unpaired >> >>>>>>>> >> >>>>>>> t >> >>>> >> >>>>> test >> >>>>>> >> >>>>>>> for comparing AvsT and so on. Any help will be really great. >> >>>>>>>> >> >>>>>>>> Hi, Mitra. I think that looks about right. You have already >> >>>>>>> >> >>>>>> performed >> >>>> >> >>>>> the unpaired t-test of AvsT (well, actually TvsA, but the p-values >> >>>>>>> >> >>>>>> will >> >>>> >> >>>>> be >> >>>>>> >> >>>>>>> the same) as coefficient 3. >> >>>>>>> >> >>>>>>> Sean >> >>>>>>> >> >>>>>>> >> >>>>>>> >> >>>>>> >> >>>>>> -- >> >>>>>> Dr. Suparna Mitra >> >>>>>> Wolfson Centre for Personalised Medicine >> >>>>>> Department of Molecular and Clinical Pharmacology >> >>>>>> Institute of Translational Medicine University of Liverpool >> >>>>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >> >>>>>> >> >>>>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >> >>>>>> M: +44 (0) 7511387895 >> >>>>>> Email id: smitra@liverpool.ac.uk >> >>>>>> Alternative Email id: suparna.mitra.sm@gmail.com >> >>>>>> >> >>>>>> [[alternative HTML version deleted]] >> >>>>>> >> >>>>>> ______________________________**_________________ >> >>>>>> Bioconductor mailing list >> >>>>>> Bioconductor@r-project.org >> >>>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< >> https://stat.ethz.ch/mailman/listinfo/bioconductor> >> >>>>>> Search the archives: >> >>>>>> >> http://news.gmane.org/gmane.**science.biology.informatics.**conductor< >> http://news.gmane.org/gmane.science.biology.informatics.conductor> >> >>>>>> >> >>>>>> [[alternative HTML version deleted]] >> >>>>> >> >>>>> ______________________________**_________________ >> >>>>> Bioconductor mailing list >> >>>>> Bioconductor@r-project.org >> >>>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< >> https://stat.ethz.ch/mailman/listinfo/bioconductor> >> >>>>> Search the archives: >> >>>>> >> http://news.gmane.org/gmane.**science.biology.informatics.**conductor< >> http://news.gmane.org/gmane.science.biology.informatics.conductor> >> >> >>>>> >> >>>>> >> >>>> >> >>>> -- >> >>>> Dr. Suparna Mitra >> >>>> Wolfson Centre for Personalised Medicine >> >>>> Department of Molecular and Clinical Pharmacology >> >>>> Institute of Translational Medicine University of Liverpool >> >>>> Block A: Waterhouse Buildings, L69 3GL Liverpool >> >>>> >> >>>> Tel. +44 (0)151 795 5394, Internal ext: 55394 >> >>>> M: +44 (0) 7511387895 >> >>>> Email id: smitra@liverpool.ac.uk >> >>>> Alternative Email id: suparna.mitra.sm@gmail.com >> >>>> >> >>>> ______________________________**_________________ >> >>>> Bioconductor mailing list >> >>>> Bioconductor@r-project.org >> >>>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< >> https://stat.ethz.ch/mailman/listinfo/bioconductor> >> >>>> Search the archives: >> >>>> >> http://news.gmane.org/gmane.**science.biology.informatics.**conductor< >> http://news.gmane.org/gmane.science.biology.informatics.conductor> >> >>>> >> >>>> [[alternative HTML version deleted]] >> >>> >> >>> ______________________________**_________________ >> >>> Bioconductor mailing list >> >>> Bioconductor@r-project.org >> >>> https://stat.ethz.ch/mailman/**listinfo/bioconductor< >> https://stat.ethz.ch/mailman/listinfo/bioconductor> >> >>> Search the archives: http://news.gmane.org/gmane.** >> >>> science.biology.informatics.**conductor< >> http://news.gmane.org/gmane.science.biology.informatics.conductor> >> >> >>> >> >> >> >> -- >> >> James W. MacDonald, M.S. >> >> Biostatistician >> >> University of Washington >> >> Environmental and Occupational Health Sciences >> >> 4225 Roosevelt Way NE, # 100 >> >> Seattle WA 98105-6099 >> >> >> >> >> > >> > >> > -- >> > Dr. Suparna Mitra >> > Wolfson Centre for Personalised Medicine >> > Department of Molecular and Clinical Pharmacology >> > Institute of Translational Medicine University of Liverpool >> > Block A: Waterhouse Buildings, L69 3GL Liverpool >> > >> > Tel. +44 (0)151 795 5394, Internal ext: 55394 >> > M: +44 (0) 7511387895 >> > Email id: smitra@liverpool.ac.uk >> > Alternative Email id: suparna.mitra.sm@gmail.com >> > >> > >> >> >> -- >> Dr. Suparna Mitra >> Wolfson Centre for Personalised Medicine >> Department of Molecular and Clinical Pharmacology >> Institute of Translational Medicine University of Liverpool >> Block A: Waterhouse Buildings, L69 3GL Liverpool >> >> Tel. +44 (0)151 795 5394, Internal ext: 55394 >> M: +44 (0) 7511387895 >> Email id: smitra@liverpool.ac.uk >> Alternative Email id: suparna.mitra.sm@gmail.com >> >> >> >> >> -- >> Dr. Suparna Mitra >> Wolfson Centre for Personalised Medicine >> Department of Molecular and Clinical Pharmacology >> Institute of Translational Medicine University of Liverpool >> Block A: Waterhouse Buildings, L69 3GL Liverpool >> >> Tel. +44 (0)151 795 5394, Internal ext: 55394 >> M: +44 (0) 7511387895 >> Email id: smitra@liverpool.ac.uk >> Alternative Email id: suparna.mitra.sm@gmail.com >> >> [[alternative HTML version deleted]] >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor@r-project.org >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >> > > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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Thanks a lot Sean, > Unfortunately, you have no significantly differentially-expressed genes. > Note that all of the adjusted p-values are pretty high. You can try to > filter your genes based on variance before testing to try to reduce the > number of genes entering your test and multiple correction. > Here I am lost again. Can you please suggest me ...is it something based on CV? However, having worked with this kind of biological system (patients), you > may suffering from a problem of a small biological effect in the setting of > large biological variation. A larger sample size may be necessary. > Yes I know, Thanks a lot for help. Mitra > > Sean > > >> Here is what I tried. >> >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") >> >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> -2.023533 >> >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >> -2.323922 >> >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >> -2.454618 >> >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >> -2.607991 >> >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >> -2.685960 >> >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >> -2.711203 >> >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >> -2.800385 >> >> > topTable(fit2.invivo, coef = 2, adjust = "fdr") >> >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 >> -2.469821 >> >> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 >> -2.652426 >> >> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 >> -2.662042 >> >> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 >> -2.702782 >> >> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 >> -2.707492 >> >> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 >> -2.717698 >> >> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 >> -2.734873 >> >> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 >> -2.772143 >> >> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 >> -2.779941 >> >> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 >> -2.785214 >> >> > topTable(fit2.invivo, coef = 3, adjust = "fdr") >> >> ID logFC AveExpr t P.Value adj.P.Val >> B >> >> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 >> -0.6982314 >> >> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 >> -1.2472882 >> >> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 >> -1.2727646 >> >> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 >> -1.2831553 >> >> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 >> -1.4031177 >> >> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 >> -1.4733549 >> >> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 >> -1.5025187 >> >> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 >> -1.5612836 >> >> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 >> -1.6535466 >> >> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 >> -1.6662493 >> >> > >> >> > >> >> > results <- decideTests(fit2.invivo) >> >> > vennDiagram(results) >> see the plot attached. >> Thanks, >> Mitra >> >> >> On 7 September 2012 12:03, Sean Davis <sdavis2@mail.nih.gov> wrote: >> >> > On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra <smitra@liverpool.ac.uk>> > >wrote: >> > >> > > Dear Sean, >> > > I have been reading Bioconductor and limma user guide and thus this >> is >> > I >> > > tried. >> > > But just being a novice, wanted to make sure if I am right. >> > > I know I have perform t-test when I created the contrast, but can you >> > > please help me how can I perform unpaired t-test here. I am concerned >> as >> > > the patients in groups are not same. >> > > >> > >> > >> > The t-test you performed was unpaired; unpaired is the "default". >> > >> > Sean >> > >> > >> > > Thanks, >> > > Mitra >> > > >> > > On 7 September 2012 11:41, Sean Davis <sdavis2@mail.nih.gov> wrote: >> > > >> > > > >> > > > >> > > > On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra < >> smitra@liverpool.ac.uk >> > > >wrote: >> > > > >> > > >> Hello Group, >> > > >> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using >> BiC. >> > > >> Previously i was using different softwares for this. And this is my >> > > first >> > > >> try with Bioconductor for big experiment. So thought to get some >> > advice >> > > in >> > > >> the beginning. >> > > >> I have Three groups of patient: (In-vivo) >> > > >> A-Acute reaction. Patient taking a drug X develops reaction. >> > > >> R-recovered (6 weeks after acute reaction-not longer taking drug >> X). >> > > >> T-Tolerant. Patient on X and tolerating treatment. >> > > >> >> > > >> Now in in-vitro study we used another constant Y >> > > >> RXY recovered and challenged with X+Y >> > > >> RY recovered challenged with only Y. RXY vs RY are to exclude >> > effects >> > > >> by >> > > >> Y. >> > > >> TXY tolerant and challenged with X+Y, >> > > >> TY tolerant challenged with only Y. TXY vs TY are to exclude >> effects >> > > by >> > > >> Y. >> > > >> >> > > >> No I want to check the cross relation and effects A vs R, RvsT and >> > Avs T >> > > >> and differentially expressed genes for each comparison. And the >> same >> > in >> > > >> invitro. There are not same patients in different groups, thus I >> > think I >> > > >> want to apply unpaired-t test. >> > > >> >> > > >> This is what I tried: >> > > >> > sessionInfo() >> > > >> R version 2.15.1 (2012-06-22) >> > > >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) >> > > >> >> > > >> locale: >> > > >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 >> > > >> >> > > >> attached base packages: >> > > >> [1] stats graphics grDevices utils datasets methods >> base >> > > >> >> > > >> other attached packages: >> > > >> [1] statmod_1.4.15 limma_3.12.1 >> > > >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 >> > > >> org.Hs.eg.db_2.7.1 >> > > >> >> > > >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 >> > > KEGG.db_2.7.1 >> > > >> GO.db_2.7.1 AnnotationDbi_1.18.1 >> > > >> [11] affy_1.34.0 Biobase_2.16.0 >> > > >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 >> > RSQLite_0.11.1 >> > > >> >> > > >> [16] DBI_0.2-5 oligo_1.20.4 >> > > >> oligoClasses_1.18.0 >> > > >> >> > > >> >> > > >> rmaOligoinvivo = oligo::rma(InVivodat1) >> > > >> Background correcting >> > > >> Normalizing >> > > >> Calculating Expression >> > > >> >> > > >> > rmaOligoinvitro = oligo::rma(InVitrodat1) >> > > >> Background correcting >> > > >> Normalizing >> > > >> Calculating Expression >> > > >> >> > > >> > maplot(rmaOligoinvivo) >> > > >> > maplot(rmaOligoinvitro) >> > > >> > InVivoTargets >> > > >> FileName Treatment >> > > >> 1 MC1 A >> > > >> 2 MC2 A >> > > >> 3 MC3 A >> > > >> 4 MC4 A >> > > >> 5 MC5 A >> > > >> 6 MC6 A >> > > >> 7 MC7 R >> > > >> 8 MC8 R >> > > >> 9 MC9 R >> > > >> 10 MC10 R >> > > >> 11 MC11 R >> > > >> 12 MC12 R >> > > >> 13 MC13 T >> > > >> 14 MC14 T >> > > >> 15 MC15 T >> > > >> 16 MC16 T >> > > >> 17 MC17 T >> > > >> 18 MC18 T >> > > >> > >> > > >> >> > > >> >> > > >> > >> InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") >> > > >> > InVitroTargets >> > > >> FileName Treatment Batch CD4 >> > > >> 1 MC19 RY 1 High >> > > >> 2 MC20 TY 1 Low >> > > >> 3 MC21 RY 2 High >> > > >> 4 MC22 TY 2 High >> > > >> 5 MC23 TY 2 Low >> > > >> 6 MC24 RY 2 High >> > > >> 7 MC25 TXY 1 Low >> > > >> 8 MC26 RXY 1 High >> > > >> 9 MC27 RXY 2 Low >> > > >> 10 MC28 TXY 2 High >> > > >> 11 MC29 RXY 2 High >> > > >> 12 MC30 TXY 2 High >> > > >> >> > > >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", >> "T")) >> > > >> >> > > >> design.invivo <- model.matrix(~0 + f.invivo) >> > > >> >> > > >> > >> > > >> >> > > >> > colnames(design.invivo) <- c("A", "R", "T") >> > > >> >> > > >> > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) >> > > >> >> > > >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = >> > > >> design.invivo) >> > > >> >> > > >> > fit2.invivo <- contrasts.fit(fit.invivo, contrast.matrix.invivo) >> > > >> >> > > >> > fit2.invivo <-eBayes(fit2.invivo) >> > > >> >> > > >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") >> > > >> >> > > >> ID logFC AveExpr t P.Value adj.P.Val >> > > >> B >> > > >> >> > > >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> > > >> -2.023533 >> > > >> >> > > >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> > > >> -2.023533 >> > > >> >> > > >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> > > >> -2.023533 >> > > >> >> > > >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 >> > > >> -2.023533 >> > > >> >> > > >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 >> > > >> -2.323922 >> > > >> >> > > >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 >> > > >> -2.454618 >> > > >> >> > > >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 >> > > >> -2.607991 >> > > >> >> > > >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 >> > > >> -2.685960 >> > > >> >> > > >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 >> > > >> -2.711203 >> > > >> >> > > >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 >> > > >> -2.800385 >> > > >> I am progressing in a right way? Further I want to perform >> unpaired t >> > > test >> > > >> for comparing AvsT and so on. Any help will be really great. >> > > >> >> > > > >> > > > Hi, Mitra. I think that looks about right. You have already >> performed >> > > > the unpaired t-test of AvsT (well, actually TvsA, but the p-values >> will >> > > be >> > > > the same) as coefficient 3. >> > > > >> > > > Sean >> > > > >> > > > >> > > >> > > >> > > >> > > -- >> > > Dr. Suparna Mitra >> > > Wolfson Centre for Personalised Medicine >> > > Department of Molecular and Clinical Pharmacology >> > > Institute of Translational Medicine University of Liverpool >> > > Block A: Waterhouse Buildings, L69 3GL Liverpool >> > > >> > > Tel. +44 (0)151 795 5394, Internal ext: 55394 >> > > M: +44 (0) 7511387895 >> > > Email id: smitra@liverpool.ac.uk >> > > Alternative Email id: suparna.mitra.sm@gmail.com >> > > >> > > [[alternative HTML version deleted]] >> > > >> > > _______________________________________________ >> > > Bioconductor mailing list >> > > Bioconductor@r-project.org >> > > https://stat.ethz.ch/mailman/listinfo/bioconductor >> > > Search the archives: >> > > http://news.gmane.org/gmane.science.biology.informatics.conductor >> > > >> > >> > [[alternative HTML version deleted]] >> > >> > _______________________________________________ >> > Bioconductor mailing list >> > Bioconductor@r-project.org >> > https://stat.ethz.ch/mailman/listinfo/bioconductor >> > Search the archives: >> > http://news.gmane.org/gmane.science.biology.informatics.conductor >> > >> >> >> >> -- >> Dr. Suparna Mitra >> Wolfson Centre for Personalised Medicine >> Department of Molecular and Clinical Pharmacology >> Institute of Translational Medicine University of Liverpool >> Block A: Waterhouse Buildings, L69 3GL Liverpool >> >> Tel. +44 (0)151 795 5394, Internal ext: 55394 >> M: +44 (0) 7511387895 >> Email id: smitra@liverpool.ac.uk >> Alternative Email id: suparna.mitra.sm@gmail.com >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor@r-project.org >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >> > > -- Dr. Suparna Mitra Wolfson Centre for Personalised Medicine Department of Molecular and Clinical Pharmacology Institute of Translational Medicine University of Liverpool Block A: Waterhouse Buildings, L69 3GL Liverpool Tel. +44 (0)151 795 5394, Internal ext: 55394 M: +44 (0) 7511387895 Email id: smitra@liverpool.ac.uk Alternative Email id: suparna.mitra.sm@gmail.com [[alternative HTML version deleted]]
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On Fri, Sep 7, 2012 at 7:33 AM, suparna mitra <smitra@liverpool.ac.uk>wrote: > Thanks a lot Sean, > > > > Unfortunately, you have no significantly differentially-expressed genes. > > Note that all of the adjusted p-values are pretty high. You can try to > > filter your genes based on variance before testing to try to reduce the > > number of genes entering your test and multiple correction. > > > Here I am lost again. Can you please suggest me ...is it something based on > CV? > Using CV or variance will likely give similar results. Basically, calculate the variance (or CV) per row of your expression values and then use the top X % (most variable) genes for limma input. Sean > > However, having worked with this kind of biological system (patients), you > > may suffering from a problem of a small biological effect in the setting > of > > large biological variation. A larger sample size may be necessary. > > > Yes I know, Thanks a lot for help. > Mitra > > > > > Sean > > > > > >> Here is what I tried. > >> > >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") > >> > >> ID logFC AveExpr t P.Value adj.P.Val > >> B > >> > >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 0.3282013 > >> -2.023533 > >> > >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 0.9552974 > >> -2.323922 > >> > >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 0.9998796 > >> -2.454618 > >> > >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 0.9998796 > >> -2.607991 > >> > >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 0.9998796 > >> -2.685960 > >> > >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 0.9998796 > >> -2.711203 > >> > >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 0.9998796 > >> -2.800385 > >> > >> > topTable(fit2.invivo, coef = 2, adjust = "fdr") > >> > >> ID logFC AveExpr t P.Value adj.P.Val > >> B > >> > >> 621 7893126 -0.5848178 4.412764 -4.577179 0.0002321630 0.9999684 > >> -2.469821 > >> > >> 6238 7917530 -0.5783362 11.170012 -4.255023 0.0004737013 0.9999684 > >> -2.652426 > >> > >> 26642 8120756 -1.0354557 5.439265 -4.238568 0.0004913467 0.9999684 > >> -2.662042 > >> > >> 1687 7894197 -0.9004303 2.631359 -4.169362 0.0005731153 0.9999684 > >> -2.702782 > >> > >> 2353 7894871 0.8441561 4.815714 4.161413 0.0005833454 0.9999684 > >> -2.707492 > >> > >> 3641 7896166 -0.6206262 7.735431 -4.144225 0.0006060986 0.9999684 > >> -2.717698 > >> > >> 2088 7894602 0.4713716 2.841855 4.115413 0.0006462632 0.9999684 > >> -2.734873 > >> > >> 5638 7911243 -0.7263053 5.676410 -4.053352 0.0007421075 0.9999684 > >> -2.772143 > >> > >> 7851 7933619 0.4194965 8.480778 4.040446 0.0007637691 0.9999684 > >> -2.779941 > >> > >> 20151 8056222 -0.8981049 7.892249 -4.031734 0.0007787485 0.9999684 > >> -2.785214 > >> > >> > topTable(fit2.invivo, coef = 3, adjust = "fdr") > >> > >> ID logFC AveExpr t P.Value adj.P.Val > >> B > >> > >> 2590 7895109 -0.9415442 4.766552 -5.803704 1.670491e-05 0.5562234 > >> -0.6982314 > >> > >> 6210 7917182 -0.2981341 3.273225 -5.028595 8.656989e-05 0.6545102 > >> -1.2472882 > >> > >> 27812 8132245 -0.4595908 5.409405 -4.995303 9.304487e-05 0.6545102 > >> -1.2727646 > >> > >> 867 7893372 1.3251627 3.017891 4.981783 9.581361e-05 0.6545102 > >> -1.2831553 > >> > >> 26802 8122099 -0.4740894 4.548920 -4.828048 1.338927e-04 0.6545102 > >> -1.4031177 > >> > >> 808 7893313 1.0125247 7.938503 4.739949 1.623493e-04 0.6545102 > >> -1.4733549 > >> > >> 26093 8115516 -0.5100673 6.294000 -4.703760 1.757561e-04 0.6545102 > >> -1.5025187 > >> > >> 587 7893092 -0.9608515 6.013864 -4.631511 2.059886e-04 0.6545102 > >> -1.5612836 > >> > >> 22913 8084605 -0.3491973 6.211757 -4.519801 2.634837e-04 0.6545102 > >> -1.6535466 > >> > >> 3828 7896353 0.6239117 4.207636 4.504578 2.724902e-04 0.6545102 > >> -1.6662493 > >> > >> > > >> > >> > > >> > >> > results <- decideTests(fit2.invivo) > >> > >> > vennDiagram(results) > >> see the plot attached. > >> Thanks, > >> Mitra > >> > >> > >> On 7 September 2012 12:03, Sean Davis <sdavis2@mail.nih.gov> wrote: > >> > >> > On Fri, Sep 7, 2012 at 6:57 AM, suparna mitra <smitra@liverpool.ac.uk> >> > >wrote: > >> > > >> > > Dear Sean, > >> > > I have been reading Bioconductor and limma user guide and thus > this > >> is > >> > I > >> > > tried. > >> > > But just being a novice, wanted to make sure if I am right. > >> > > I know I have perform t-test when I created the contrast, but can > you > >> > > please help me how can I perform unpaired t-test here. I am > concerned > >> as > >> > > the patients in groups are not same. > >> > > > >> > > >> > > >> > The t-test you performed was unpaired; unpaired is the "default". > >> > > >> > Sean > >> > > >> > > >> > > Thanks, > >> > > Mitra > >> > > > >> > > On 7 September 2012 11:41, Sean Davis <sdavis2@mail.nih.gov> wrote: > >> > > > >> > > > > >> > > > > >> > > > On Fri, Sep 7, 2012 at 5:54 AM, suparna mitra < > >> smitra@liverpool.ac.uk > >> > > >wrote: > >> > > > > >> > > >> Hello Group, > >> > > >> I am trying t analyze my affymetrix (HuGene-1_0-st-v1) data using > >> BiC. > >> > > >> Previously i was using different softwares for this. And this is > my > >> > > first > >> > > >> try with Bioconductor for big experiment. So thought to get some > >> > advice > >> > > in > >> > > >> the beginning. > >> > > >> I have Three groups of patient: (In-vivo) > >> > > >> A-Acute reaction. Patient taking a drug X develops reaction. > >> > > >> R-recovered (6 weeks after acute reaction-not longer taking drug > >> X). > >> > > >> T-Tolerant. Patient on X and tolerating treatment. > >> > > >> > >> > > >> Now in in-vitro study we used another constant Y > >> > > >> RXY recovered and challenged with X+Y > >> > > >> RY recovered challenged with only Y. RXY vs RY are to exclude > >> > effects > >> > > >> by > >> > > >> Y. > >> > > >> TXY tolerant and challenged with X+Y, > >> > > >> TY tolerant challenged with only Y. TXY vs TY are to exclude > >> effects > >> > > by > >> > > >> Y. > >> > > >> > >> > > >> No I want to check the cross relation and effects A vs R, RvsT > and > >> > Avs T > >> > > >> and differentially expressed genes for each comparison. And the > >> same > >> > in > >> > > >> invitro. There are not same patients in different groups, thus I > >> > think I > >> > > >> want to apply unpaired-t test. > >> > > >> > >> > > >> This is what I tried: > >> > > >> > sessionInfo() > >> > > >> R version 2.15.1 (2012-06-22) > >> > > >> Platform: i386-apple-darwin9.8.0/i386 (32-bit) > >> > > >> > >> > > >> locale: > >> > > >> [1] en_GB.UTF-8/en_GB.UTF-8/en_GB.UTF-8/C/en_GB.UTF-8/en_GB.UTF-8 > >> > > >> > >> > > >> attached base packages: > >> > > >> [1] stats graphics grDevices utils datasets methods > >> base > >> > > >> > >> > > >> other attached packages: > >> > > >> [1] statmod_1.4.15 limma_3.12.1 > >> > > >> annotate_1.34.1 hugene10stprobeset.db_8.0.1 > >> > > >> org.Hs.eg.db_2.7.1 > >> > > >> > >> > > >> [6] BiocInstaller_1.4.7 affycoretools_1.28.0 > >> > > KEGG.db_2.7.1 > >> > > >> GO.db_2.7.1 AnnotationDbi_1.18.1 > >> > > >> [11] affy_1.34.0 Biobase_2.16.0 > >> > > >> BiocGenerics_0.2.0 pd.hugene.1.0.st.v1_3.6.0 > >> > RSQLite_0.11.1 > >> > > >> > >> > > >> [16] DBI_0.2-5 oligo_1.20.4 > >> > > >> oligoClasses_1.18.0 > >> > > >> > >> > > >> > >> > > >> rmaOligoinvivo = oligo::rma(InVivodat1) > >> > > >> Background correcting > >> > > >> Normalizing > >> > > >> Calculating Expression > >> > > >> > >> > > >> > rmaOligoinvitro = oligo::rma(InVitrodat1) > >> > > >> Background correcting > >> > > >> Normalizing > >> > > >> Calculating Expression > >> > > >> > >> > > >> > maplot(rmaOligoinvivo) > >> > > >> > maplot(rmaOligoinvitro) > >> > > >> > InVivoTargets > >> > > >> FileName Treatment > >> > > >> 1 MC1 A > >> > > >> 2 MC2 A > >> > > >> 3 MC3 A > >> > > >> 4 MC4 A > >> > > >> 5 MC5 A > >> > > >> 6 MC6 A > >> > > >> 7 MC7 R > >> > > >> 8 MC8 R > >> > > >> 9 MC9 R > >> > > >> 10 MC10 R > >> > > >> 11 MC11 R > >> > > >> 12 MC12 R > >> > > >> 13 MC13 T > >> > > >> 14 MC14 T > >> > > >> 15 MC15 T > >> > > >> 16 MC16 T > >> > > >> 17 MC17 T > >> > > >> 18 MC18 T > >> > > >> > > >> > > >> > >> > > >> > >> > > > >> > > >> > InVitroTargets=readTargets("~/Desktop/Recent/Liverpool-work- related/Micro_RawData/InVitroTargets.txt") > >> > > >> > InVitroTargets > >> > > >> FileName Treatment Batch CD4 > >> > > >> 1 MC19 RY 1 High > >> > > >> 2 MC20 TY 1 Low > >> > > >> 3 MC21 RY 2 High > >> > > >> 4 MC22 TY 2 High > >> > > >> 5 MC23 TY 2 Low > >> > > >> 6 MC24 RY 2 High > >> > > >> 7 MC25 TXY 1 Low > >> > > >> 8 MC26 RXY 1 High > >> > > >> 9 MC27 RXY 2 Low > >> > > >> 10 MC28 TXY 2 High > >> > > >> 11 MC29 RXY 2 High > >> > > >> 12 MC30 TXY 2 High > >> > > >> > >> > > >> f.invivo <- factor(InVivoTargets$Treatment, levels = c("A", "R", > >> "T")) > >> > > >> > >> > > >> design.invivo <- model.matrix(~0 + f.invivo) > >> > > >> > >> > > >> > > >> > > >> > >> > > >> > colnames(design.invivo) <- c("A", "R", "T") > >> > > >> > >> > > >> > fit.invivo <- lmFit(rmaOligoinvivo, design.invivo) > >> > > >> > >> > > >> > contrast.matrix.invivo <- makeContrasts(R-A, T-R, T-A,levels = > >> > > >> design.invivo) > >> > > >> > >> > > >> > fit2.invivo <- contrasts.fit(fit.invivo, > contrast.matrix.invivo) > >> > > >> > >> > > >> > fit2.invivo <-eBayes(fit2.invivo) > >> > > >> > >> > > >> > topTable(fit2.invivo, coef = 1, adjust = "fdr") > >> > > >> > >> > > >> ID logFC AveExpr t P.Value > adj.P.Val > >> > > >> B > >> > > >> > >> > > >> 8819 7943047 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >> > > >> -2.023533 > >> > > >> > >> > > >> 9675 7950951 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >> > > >> -2.023533 > >> > > >> > >> > > >> 18889 8043581 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >> > > >> -2.023533 > >> > > >> > >> > > >> 19899 8053785 -0.3640702 4.177681 -5.395110 3.942713e-05 > 0.3282013 > >> > > >> -2.023533 > >> > > >> > >> > > >> 3713 7896238 0.7731154 2.999029 4.796490 1.434510e-04 > 0.9552974 > >> > > >> -2.323922 > >> > > >> > >> > > >> 19926 8054075 -0.3816217 4.062936 -4.557543 2.424324e-04 > 0.9998796 > >> > > >> -2.454618 > >> > > >> > >> > > >> 18660 8041642 -1.0007299 4.220083 -4.290346 4.379518e-04 > 0.9998796 > >> > > >> -2.607991 > >> > > >> > >> > > >> 3759 7896284 -0.7555604 5.727302 -4.159251 5.861601e-04 > 0.9998796 > >> > > >> -2.685960 > >> > > >> > >> > > >> 6238 7917530 0.5596335 11.170012 4.117421 6.433789e-04 > 0.9998796 > >> > > >> -2.711203 > >> > > >> > >> > > >> 15545 8010622 -0.3324189 3.771856 -3.971869 8.899739e-04 > 0.9998796 > >> > > >> -2.800385 > >> > > >> I am progressing in a right way? Further I want to perform > >> unpaired t > >> > > test > >> > > >> for comparing AvsT and so on. Any help will be really great. > >> > > >> > >> > > > > >> > > > Hi, Mitra. I think that looks about right. You have already > >> performed > >> > > > the unpaired t-test of AvsT (well, actually TvsA, but the p-values > >> will > >> > > be > >> > > > the same) as coefficient 3. > >> > > > > >> > > > Sean > >> > > > > >> > > > > >> > > > >> > > > >> > > > >> > > -- > >> > > Dr. Suparna Mitra > >> > > Wolfson Centre for Personalised Medicine > >> > > Department of Molecular and Clinical Pharmacology > >> > > Institute of Translational Medicine University of Liverpool > >> > > Block A: Waterhouse Buildings, L69 3GL Liverpool > >> > > > >> > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > >> > > M: +44 (0) 7511387895 > >> > > Email id: smitra@liverpool.ac.uk > >> > > Alternative Email id: suparna.mitra.sm@gmail.com > >> > > > >> > > [[alternative HTML version deleted]] > >> > > > >> > > _______________________________________________ > >> > > Bioconductor mailing list > >> > > Bioconductor@r-project.org > >> > > https://stat.ethz.ch/mailman/listinfo/bioconductor > >> > > Search the archives: > >> > > http://news.gmane.org/gmane.science.biology.informatics.conductor > >> > > > >> > > >> > [[alternative HTML version deleted]] > >> > > >> > _______________________________________________ > >> > Bioconductor mailing list > >> > Bioconductor@r-project.org > >> > https://stat.ethz.ch/mailman/listinfo/bioconductor > >> > Search the archives: > >> > http://news.gmane.org/gmane.science.biology.informatics.conductor > >> > > >> > >> > >> > >> -- > >> Dr. Suparna Mitra > >> Wolfson Centre for Personalised Medicine > >> Department of Molecular and Clinical Pharmacology > >> Institute of Translational Medicine University of Liverpool > >> Block A: Waterhouse Buildings, L69 3GL Liverpool > >> > >> Tel. +44 (0)151 795 5394, Internal ext: 55394 > >> M: +44 (0) 7511387895 > >> Email id: smitra@liverpool.ac.uk > >> Alternative Email id: suparna.mitra.sm@gmail.com > >> > >> _______________________________________________ > >> Bioconductor mailing list > >> Bioconductor@r-project.org > >> https://stat.ethz.ch/mailman/listinfo/bioconductor > >> Search the archives: > >> http://news.gmane.org/gmane.science.biology.informatics.conductor > >> > > > > > > > -- > Dr. Suparna Mitra > Wolfson Centre for Personalised Medicine > Department of Molecular and Clinical Pharmacology > Institute of Translational Medicine University of Liverpool > Block A: Waterhouse Buildings, L69 3GL Liverpool > > Tel. +44 (0)151 795 5394, Internal ext: 55394 > M: +44 (0) 7511387895 > Email id: smitra@liverpool.ac.uk > Alternative Email id: suparna.mitra.sm@gmail.com > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor@r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]
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