[BioC] warnings or potential problems in limma procedure

Yi, Ming (NIH/NCI) [C] yiming at mail.nih.gov
Wed Apr 13 15:31:45 CEST 2011


Hi, Dear List:

I am still looking for some explanation or diagnosis about the following potential issue that I am not sure what I did is wrong or fine (I apologize if my previous post is not quite clear to the list)


I am using limma to do the paired test on the following setting, my tar object looks like as below:

> tar[1:5,]
   AccNum Patient_Type_Comb Type RACE  ER
67 S10184    S10184_N_W_NEG    N    W NEG
66 S10184    S10184_T_W_NEG    T    W NEG
68 S10330    S10330_N_B_NEG    N    B NEG
69 S10330    S10330_T_B_NEG    T    B NEG
74 S10601    S10601_N_W_POS    N    W POS

AccNum is the patient ID and the same patient have two types of samples: "N" for normal, "T" for tumor,

Two Races in the sample population: W for "White", B for "Black"

ER is for ER status: NEG for negative, POS for positive

Patient_Type_Comb column is for showing the sample phenotype in one string

The goal of the analysis is looking for the differential gene lists for each of the contrasts including ER positive tumor vs ER positive normal for matched same patient of only Black population (Africa America population), ER negative tumor vs ER negative normal for matched same patients of only White population (Caucasian population) etc as you can see more details in my design and contrast matrix setting (the key is need to consider the paired samples for the same patient with both tumor and normal samples (normal is surrounding normal tissue of the tumor tissue for the same patient), which is well controlled study.


My data matrix (Partial, Array data) looks like the following:

> mydata[1:5,1:4]
        S10184_N_W_NEG S10184_T_W_NEG S10330_N_B_NEG S10330_T_B_NEG
7936596      10.079810      10.810695      10.733401      11.369506
8037331      10.076718      10.217359      10.921994      10.389894
8023672       8.503989       8.786565       8.936260       9.384205
8128282       5.423744       4.826185       5.872070       4.486140
8063634       5.909231       6.773356       6.653584       6.408861

Here is how I set up my design and contrast matrix:

>group1<-paste(tar$RACE,tar$Type,tar$ER, sep=".");
> unique(group1)
[1] "W.N.NEG" "W.T.NEG" "B.N.NEG" "B.T.NEG" "W.N.POS" "W.T.POS" "B.N.POS" "B.T.POS"
> group<-factor(group1, levels=c(  "W.N.NEG","W.T.NEG", "B.N.NEG", 
>"B.T.NEG", "W.N.POS", "W.T.POS", "B.N.POS", "B.T.POS")) 
>Samples<-factor(tar$AccNum);

design<-model.matrix(~-1+group+Samples);
> colnames(design)<-sub("group","",colnames(design));
> colnames(design)<-sub("Samples","",colnames(design));
> con.matrix<-makeContrasts(T.POS_N.POS=B.T.POS+W.T.POS-B.N.POS-W.N.POS,
+ B.T.POS_B.N.POS=B.T.POS-B.N.POS,W.T.POS_W.N.POS=W.T.POS-W.N.POS,
+ T.NEG_N.NEG=B.T.NEG+W.T.NEG-B.N.NEG-W.N.NEG,
+ B.T.NEG_B.N.NEG=B.T.NEG-B.N.NEG, W.T.NEG_W.N.NEG=W.T.NEG-W.N.NEG,
+ levels=design)


Here is the partical contrast matrix:
> con.matrix[,]
         Contrasts
Levels    T.POS_N.POS B.T.POS_B.N.POS W.T.POS_W.N.POS T.NEG_N.NEG B.T.NEG_B.N.NEG W.T.NEG_W.N.NEG
  W.N.NEG           0               0               0          -1               0              -1
  W.T.NEG           0               0               0           1               0               1
  B.N.NEG           0               0               0          -1              -1               0
  B.T.NEG           0               0               0           1               1               0
  W.N.POS          -1               0              -1           0               0               0
  W.T.POS           1               0               1           0               0               0
  B.N.POS          -1              -1               0           0               0               0
  B.T.POS           1               1               0           0               0               0
  S10330            0               0               0           0               0               0
  S10601            0               0               0           0               0               0
  S10618            0               0               0           0               0               0
  S10929            0               0               0           0               0               0
  S10940            0               0               0           0               0               0


However, when I tried to fir the data into the limma model, I run into the following warnings, which is what I am trying asking about:

> lmFit(mydata,design)->fit1;
Coefficients not estimable: S14697 S14730 S14810 Warning message:
Partial NA coefficients for 26804 probe(s)

This warning seems not bothering the subsequent steps as shown below, but I am not sure why I get warning here, could the list provide some insights or clues for me? that would be highly appreciated!

>contrasts.fit(fit1, con.matrix)->fit2
> eBayes(fit2)->fit3
> allContrast<-colnames(fit3);
> allContrast
[1] "T.POS_N.POS"     "B.T.POS_B.N.POS" "W.T.POS_W.N.POS" "T.NEG_N.NEG"     "B.T.NEG_B.N.NEG" "W.T.NEG_W.N.NEG"

I also did check specifically for the samples listed in the warning message

> tar[tar$AccNum %in% c("S14697", "S14730", "S14810"),]
   AccNum Patient_Type_Comb Type RACE  ER
57 S14697    S14697_N_W_POS    N    W POS
58 S14697    S14697_T_W_POS    T    W POS
55 S14730    S14730_N_B_NEG    N    B NEG
56 S14730    S14730_T_B_NEG    T    B NEG
59 S14810    S14810_N_B_POS    N    B POS
60 S14810    S14810_T_B_POS    T    B POS

They appear to be common, which of all have paired samples (T vs N) and some of which are white/black and some are ER Negative and positive, seems not fall into any of the special category of the phenotype.

I also check specifically for their data as below:

> mydata[1:5,c("S14697_N_W_POS", "S14697_T_W_POS", "S14730_N_B_NEG", "S14730_T_B_NEG", "S14810_N_B_POS", "S14810_T_B_POS")]
        S14697_N_W_POS S14697_T_W_POS S14730_N_B_NEG S14730_T_B_NEG S14810_N_B_POS S14810_T_B_POS
7936596      11.024855      10.954703      10.832579      10.917364      10.631019      10.842098
8037331       9.807050      10.366058      10.285187       9.955208      10.410920      10.620751
8023672       8.734080       8.359230       8.559288       8.245623       8.613978       8.614790
8128282       5.489218       5.703427       5.026220       4.738774       5.362589       5.193500
8063634       6.562237       6.784427       6.632752       6.757525       6.887120       7.095357


Which also look normal to me.

Thanks a lot in advance for your advice and suggestion!

Best

Myi



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