[BioC] limma warning: Coefficients not estimable

Karl Brand k.brand at erasmusmc.nl
Wed Feb 10 17:08:34 CET 2010


Dear BioC,

Using limma, when fitting the model:
model.matrix(~Tissue * Pperiod + Time + Animal)

I get this warning:
 > fit <- lmFit(rma.pp, design)
Coefficients not estimable: Animal32 Animal33 Animal34 Animal35 Animal36 
Animal37 Animal38 Animal39 Animal40 Animal41 Animal42 Animal43 Animal44 
Animal45 Animal46 Animal47 Animal48
Warning message:
Partial NA coefficients for 45101 probe(s)

In addition, the reuslting number or DE genes for my contrasts of 
interest (which are different than the 'not estimable' ones listed in 
teh warning above) are mcuh lower than expected; & furthermore, the 
contrast-coefficents (log2FCs) and simply wrong.

When fitting a similar model, merely lacking the 'pairing' factor 
("Animal"):
model.matrix(~Tissue * Pperiod + Time)

I don't get this error. My question:

Is it me? Or am i attempting the impossible, ie., by including a factor 
for pairing (Animal) trying to fit more factors than my measurements can 
support and this is limma's way of telling me? Raw script and targets 
file below.

I really hope an experienced limma user can enlighten me on this, or 
point me to a resource suitable for a biologists level of understanding.

Thanks in advance,

Karl


 > targets <- read.delim("RNA_Targets.txt")

 > Tissue <- factor(targets$Tissue, levels = c("R", "C"))

 > Pperiod <- factor(targets$Pperiod, levels = c("E", "L", "S"))

 > Time <- factor(targets$Time, levels = c("1", "2", "3", "4",
+                                         "5", "6", "7", "8",
+                        .... [TRUNCATED]

 > Animal <- factor(targets$Animal, levels = c("1", "2", "3", "4",
+                                             "5", "6", "7", "8",
+                .... [TRUNCATED]

 > design <- model.matrix(~Tissue * Pperiod + Time + Animal)

 > colnames(design)
  [1] "(Intercept)"      "TissueC"          "PperiodL" 
"PperiodS"         "Time2"            "Time3"            "Time4" 
     "Time5"            "Time6"            "Time7"
[11] "Time8"            "Time9"            "Time10"           "Time11" 
          "Time12"           "Time13"           "Time14" 
"Time15"           "Time16"           "Animal2"
[21] "Animal3"          "Animal4"          "Animal5"          "Animal6" 
          "Animal7"          "Animal8"          "Animal9" 
"Animal10"         "Animal11"         "Animal12"
[31] "Animal13"         "Animal14"         "Animal15"         "Animal16" 
         "Animal17"         "Animal18"         "Animal19" 
"Animal20"         "Animal21"         "Animal22"
[41] "Animal23"         "Animal24"         "Animal25"         "Animal26" 
         "Animal27"         "Animal28"         "Animal29" 
"Animal30"         "Animal31"         "Animal32"
[51] "Animal33"         "Animal34"         "Animal35"         "Animal36" 
         "Animal37"         "Animal38"         "Animal39" 
"Animal40"         "Animal41"         "Animal42"
[61] "Animal43"         "Animal44"         "Animal45"         "Animal46" 
         "Animal47"         "Animal48"         "TissueC:PperiodL" 
"TissueC:PperiodS"
 > source(.trPaths[5], echo=TRUE, max.deparse.length=150)

 > fit <- lmFit(rma.pp, design)
Coefficients not estimable: Animal32 Animal33 Animal34 Animal35 Animal36 
Animal37 Animal38 Animal39 Animal40 Animal41 Animal42 Animal43 Animal44 
Animal45 Animal46 Animal47 Animal48
Warning message:
Partial NA coefficients for 45101 probe(s)
 >


FileName	Tissue	Pperiod	Time	Animal
01-PPL3-sample02.CEL	R	S	1	1
02-PPL3-sample03.CEL	C	S	1	1
03-PPL5-sample02.CEL	R	S	2	2
04-PPL5-sample03.CEL	C	S	2	2
05-PPL3-sample04.CEL	R	S	3	3
06-PPL3-sample05.CEL	C	S	3	3
07-PPL5-sample04.CEL	R	S	4	4
08-PPL5-sample05.CEL	C	S	4	4
09-PPL3-sample06.CEL	R	S	5	5
10-PPL3-sample07.CEL	C	S	5	5
11-PPL5-sample06.CEL	R	S	6	6
12-PPL5-sample07.CEL	C	S	6	6
13-PPL3-sample08.CEL	R	S	7	7
14-PPL3-sample09.CEL	C	S	7	7
15-PPL5-sample08.CEL	R	S	8	8
16-PPL5-sample09.CEL	C	S	8	8
17-PPL3-sample10.CEL	R	S	9	9
18-PPL3-sample11.CEL	C	S	9	9
19-PPL5-sample10.CEL	R	S	10	10
20-PPL5-sample11.CEL	C	S	10	10
21-PPL3-sample12.CEL	R	S	11	11
22-PPL3-sample13.CEL	C	S	11	11
23-PPL5-sample12.CEL	R	S	12	12
24-PPL5-sample13.CEL	C	S	12	12
25-PPL3-sample14.CEL	R	S	13	13
26-PPL3-sample15.CEL	C	S	13	13
27-PPL5-sample14.CEL	R	S	14	14
28-PPL5-sample15.CEL	C	S	14	14
29-PPL3-sample16.CEL	R	S	15	15
30-PPL3-sample17.CEL	C	S	15	15
31-PPL5-sample16.CEL	R	S	16	16
32-PPL5-sample17.CEL	C	S	16	16
33-PPL1-sample02.CEL	R	E	1	17
34-PPL1-sample03.CEL	C	E	1	17
35-PPL6-sample02.CEL	R	E	2	18
36-PPL6-sample03.CEL	C	E	2	18
37-PPL1-sample04.CEL	R	E	3	19
38-PPL1-sample05.CEL	C	E	3	19
39-PPL6-sample04.CEL	R	E	4	20
40-PPL6-sample05.CEL	C	E	4	20
41-PPL1-sample06.CEL	R	E	5	21
42-PPL1-sample07.CEL	C	E	5	21
43-PPL6-sample06.CEL	R	E	6	22
44-PPL6-sample07.CEL	C	E	6	22
45-PPL1-sample08.CEL	R	E	7	23
46-PPL1-sample09.CEL	C	E	7	23
47-PPL6-sample08.CEL	R	E	8	24
48-PPL6-sample09.CEL	C	E	8	24
49-PPL1-sample10.CEL	R	E	9	25
50-PPL1-sample11.CEL	C	E	9	25
51-PPL6-sample10.CEL	R	E	10	26
52-PPL6-sample11.CEL	C	E	10	26
53-PPL1-sample12.CEL	R	E	11	27
54-PPL1-sample13.CEL	C	E	11	27
55-PPL6-sample12.CEL	R	E	12	28
56-PPL6-sample13.CEL	C	E	12	28
57-PPL1-sample14.CEL	R	E	13	29
58-PPL1-sample15.CEL	C	E	13	29
59-PPL6-sample14.CEL	R	E	14	30
60-PPL6-sample15.CEL	C	E	14	30
61-PPL1-sample16.CEL	R	E	15	31
62-PPL1-sample17.CEL	C	E	15	31
63-PPL6-sample16.CEL	R	E	16	32
64-PPL6-sample17.CEL	C	E	16	32
65-PPL2-sample02.CEL	R	L	1	33
66-PPL2-sample03.CEL	C	L	1	33
67-PPL4-sample02.CEL	R	L	2	34
68-PPL4-sample03.CEL	C	L	2	34
69-PPL2-sample04.CEL	R	L	3	35
70-PPL2-sample05.CEL	C	L	3	35
71-PPL4-sample04.CEL	R	L	4	36
72-PPL4-sample05.CEL	C	L	4	36
73-PPL2-sample06.CEL	R	L	5	37
74-PPL2-sample07.CEL	C	L	5	37
75-PPL4-sample06.CEL	R	L	6	38
76-PPL4-sample07.CEL	C	L	6	38
77-PPL2-sample08.CEL	R	L	7	39
78-PPL2-sample09.CEL	C	L	7	39
79-PPL4-sample08.CEL	R	L	8	40
80-PPL4-sample09.CEL	C	L	8	40
81-PPL2-sample10.CEL	R	L	9	41
82-PPL2-sample11.CEL	C	L	9	41
83-PPL4-sample10.CEL	R	L	10	42
84-PPL4-sample11.CEL	C	L	10	42
85-PPL2-sample12.CEL	R	L	11	43
86-PPL2-sample13.CEL	C	L	11	43
87-PPL4-sample12.CEL	R	L	12	44
88-PPL4-sample13.CEL	C	L	12	44
89-PPL2-sample14.CEL	R	L	13	45
90-PPL2-sample15.CEL	C	L	13	45
91-PPL4-sample14.CEL	R	L	14	46
92-PPL4-sample15.CEL	C	L	14	46
93-PPL2-sample16.CEL	R	L	15	47
94-PPL2-sample17.CEL	C	L	15	47
95-PPL4-sample16.CEL	R	L	16	48
96-PPL4-sample17.CEL	C	L	16	48

-- 
Karl Brand <k.brand at erasmusmc.nl>
Department of Genetics
Erasmus MC
Dr Molewaterplein 50
3015 GE Rotterdam
lab +31 (0)10 704 3409 fax +31 (0)10 704 4743 mob +31 (0)642 777 268



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