Dear Lists, I'm currently using LIMMA package to analyze my microarray datasets. For each treatment, I have three biological replicates. The subset of the constructed design look like this: Array R16A R16B R16C R16D R16E S03A ------ Biorep1 Biorep2 Biorep3 02152012_462157_A01_1613_cy5_R16E 0 0 0 0 1 0 ------ 1 0 0 02152012_462157_A01_161_cy3_R16A 1 0 0 0 0 0 ------ 1 0 0 02152012_462157_A02_81_cy5_R08A 0 0 0 0 0 0 ------ 0 0 02152012_462157_A02_813_cy3_R08E 0 0 0 0 0 0 ------ 1 0 0 02152012_462157_A03_164_cy5_R16B 0 1 0 0 0 0 ------ 1 0 0 02152012_462157_A03_1614_cy3_R16E 0 0 0 0 1 0 ------ 0 1 0 02152012_462157_A04_89_cy5_R08C 0 0 0 0 0 0 0 ------ 0 1 02152012_462157_A04_811_cy3_R08D 0 0 0 0 0 0 ------ 0 1 0 02152012_462157_A05_1611_cy5_R16D 0 0 0 1 0 0 ------ 0 1 0 When I fit my data into lmFit, it doesn't estimate the coefficient of the third biological replicates. The error msg looks like this: > Fit=lmFit(norm.dataM,designlm,correlation=corfit$consensus) Coefficients not estimable: Biorep3 Warning message: Partial NA coefficients for 22664 probe(s) Can anyone tell me what's goes wrong my my design? Thanks a lot

On Fri, Mar 30, 2012 at 8:09 AM, yeeling chong <yeelingchong at="" gmail.com=""> wrote: > Dear Lists, > > I'm currently using LIMMA package to analyze my microarray datasets. > For each treatment, I have three biological replicates. The subset of > the constructed design look like this: > Array > R16A ? ?R16B ? ?R16C ? ?R16D ? ?R16E ? ?S03A ? ? ?------ ? Biorep1 ? ? ?Biorep2 Biorep3 > 02152012_462157_A01_1613_cy5_R16E ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 1 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 > 02152012_462157_A01_161_cy3_R16A ? ? ? ?1 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 > 02152012_462157_A02_81_cy5_R08A ? ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?0 > 02152012_462157_A02_813_cy3_R08E ? ? ? ?0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 > 02152012_462157_A03_164_cy5_R16B ? ? ? ?0 ? ? ? 1 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 > 02152012_462157_A03_1614_cy3_R16E ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 1 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 ? ? ? 0 > 02152012_462157_A04_89_cy5_R08C 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 > 02152012_462157_A04_811_cy3_R08D ? ? ? ?0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 ? ? ? 0 > 02152012_462157_A05_1611_cy5_R16D ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 1 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 ? ? ? 0 > > When I fit my data into lmFit, it doesn't estimate the coefficient of > the third biological replicates. The error msg looks like this: > >> Fit=lmFit(norm.dataM,designlm,correlation=corfit$consensus) > Coefficients not estimable: Biorep3 > Warning message: > Partial NA coefficients for 22664 probe(s) The coefficients not estimable means that you do not have enough degrees of freedom given your number of samples and number of effects you are trying to estimate. In the normal use case, one does not include the BiorepX columns in the design matrix. I'd suggest removing the Bioreps columns from the design matrix (unless you really are interested in the biological replicates). Sean > Can anyone tell me what's goes wrong my my design? Thanks a lot > > _______________________________________________ > 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

On Fri, Mar 30, 2012 at 8:24 AM, yeeling chong <yeelingchong at="" gmail.com=""> wrote: > Thanks Sean, > > ?I tried to remove the third replicate before and it can estimate both > biorep1 & 2 coefficients. How does this correlate with the degree of > freedom then? Thanks again. Removing a parameter from the fit increases the DOF by 1. http://en.wikipedia.org/wiki/Degrees_of_freedom_(statistics) Sean > > On Fri, Mar 30, 2012 at 8:17 AM, Sean Davis <sdavis2 at="" mail.nih.gov=""> wrote: >> On Fri, Mar 30, 2012 at 8:09 AM, yeeling chong <yeelingchong at="" gmail.com=""> wrote: >>> Dear Lists, >>> >>> I'm currently using LIMMA package to analyze my microarray datasets. >>> For each treatment, I have three biological replicates. The subset of >>> the constructed design look like this: >>> Array >>> R16A ? ?R16B ? ?R16C ? ?R16D ? ?R16E ? ?S03A ? ? ?------ ? Biorep1 ? ? ?Biorep2 Biorep3 >>> 02152012_462157_A01_1613_cy5_R16E ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 1 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 >>> 02152012_462157_A01_161_cy3_R16A ? ? ? ?1 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 >>> 02152012_462157_A02_81_cy5_R08A ? ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?0 >>> 02152012_462157_A02_813_cy3_R08E ? ? ? ?0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 >>> 02152012_462157_A03_164_cy5_R16B ? ? ? ?0 ? ? ? 1 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 1 ? ? ? ?0 ? ? ? 0 >>> 02152012_462157_A03_1614_cy3_R16E ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 1 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 ? ? ? 0 >>> 02152012_462157_A04_89_cy5_R08C 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 >>> 02152012_462157_A04_811_cy3_R08D ? ? ? ?0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 ? ? ? 0 >>> 02152012_462157_A05_1611_cy5_R16D ? ? ? 0 ? ? ? 0 ? ? ? 0 ? ? ? 1 ? ? ? 0 ? ? ? 0 ? ? ? ------ 0 ? ? ? ?1 ? ? ? 0 >>> >>> When I fit my data into lmFit, it doesn't estimate the coefficient of >>> the third biological replicates. The error msg looks like this: >>> >>>> Fit=lmFit(norm.dataM,designlm,correlation=corfit$consensus) >>> Coefficients not estimable: Biorep3 >>> Warning message: >>> Partial NA coefficients for 22664 probe(s) >> >> The coefficients not estimable means that you do not have enough >> degrees of freedom given your number of samples and number of effects >> you are trying to estimate. ?In the normal use case, one does not >> include the BiorepX columns in the design matrix. ?I'd suggest >> removing the Bioreps columns from the design matrix (unless you really >> are interested in the biological replicates). >> >> Sean >> >> >> >>> Can anyone tell me what's goes wrong my my design? Thanks a lot >>> >>> _______________________________________________ >>> 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