[BioC] edgeR: design matrix for different condition
Gordon K Smyth
smyth at wehi.EDU.AU
Mon Aug 6 09:11:21 CEST 2012
Dear KJ Lim,
Well, your "tree" column has 16 different entries, a different entry for
every library. Naturally this causes a problem.
So either you have no replication of any experimental condition, or you
have mistakingly used different labels for the same condition.
In your earlier email below, you claimed to have just two genotypes
(tree=HS and tree=LS), but in your latest targets file you have 16
different genotypes. I'm guessing that the genotypes are actually H and
L, so the entries in the targets file should be just H and L.
That's as much advice as I have time to give you, and I hope that others
will help you further. I feel that I've given you good general advice
that is applicable to your experiment.
Best wishes
Gordon
---------------------------------------------
Professor Gordon K Smyth,
Bioinformatics Division,
Walter and Eliza Hall Institute of Medical Research,
1G Royal Parade, Parkville, Vic 3052, Australia.
Tel: (03) 9345 2326, Fax (03) 9347 0852,
http://www.statsci.org/smyth
On Mon, 6 Aug 2012, KJ Lim wrote:
> Dear Prof Gordon,
>
> Good day. Thanks for you prompt replied.
>
> Below is the output of cbind(targets,Group=Group)
>
>> cbind(targets,Group=Group)
> files treat tree X Group
> 1 ./rawData/HS0H01.txt 00H H1 NA 00H.H1
> 2 ./rawData/HS0H02.txt 00H H2 NA 00H.H2
> 3 ./rawData/HS3H01.txt 03H H3 NA 03H.H3
> 4 ./rawData/HS3H02.txt 03H H4 NA 03H.H4
> 5 ./rawData/HS1D01.txt 24H H5 NA 24H.H5
> 6 ./rawData/HS1D02.txt 24H H6 NA 24H.H6
> 7 ./rawData/HS4D01.txt 96H H7 NA 96H.H7
> 8 ./rawData/HS4D02.txt 96H H8 NA 96H.H8
> 9 ./rawData/LS0H01.txt 00H L1 NA 00H.L1
> 10 ./rawData/LS0H02.txt 00H L2 NA 00H.L2
> 11 ./rawData/LS3H01.txt 03H L3 NA 03H.L3
> 12 ./rawData/LS3H02.txt 03H L4 NA 03H.L4
> 13 ./rawData/LS1D01.txt 24H L5 NA 24H.L5
> 14 ./rawData/LS1D02.txt 24H L6 NA 24H.L6
> 15 ./rawData/LS4D01.txt 96H L7 NA 96H.L7
> 16 ./rawData/LS4D02.txt 96H L8 NA 96H.L8
>
> Thank you for your time.
>
> Best regards,
> KJ Lim
>
>
> On 6 August 2012 07:33, Gordon K Smyth <smyth at wehi.edu.au> wrote:
>> The error message is pretty self-explanatory. Apparently your design matrix
>> has as many columns as there are libraries, so there are no degrees of
>> freedom left from which to estimate variability. According to the code and
>> output you have given, this message should be impossible, so you must have
>> changed the data in some way that I cannot see.
>>
>> If you had shown the output from cbind(targets,Group=Group), then I might
>> have been able to say something useful.
>>
>> Best wishes
>> Gordon
>>
>>
>> On Mon, 6 Aug 2012, KJ Lim wrote:
>>
>>> Dear Prof Gordon,
>>>
>>> Good day.
>>>
>>> Thanks for your time to update the edgeR User's Guide. It is useful.
>>>
>>> I combined all my experiment factors into one combined factor and
>>> described the experiment matrix like the example in the User's guide:
>>>
>>> > Group <- factor(paste(targets$treat, targets$tree,sep="."))
>>> > cbind(targets,Group=Group)
>>> > hl.design <- model.matrix(~0+Group)
>>>
>>> But, I encountered an error when I performed the estimate dispersion
>>> for my data
>>>
>>> > hl <- estimateGLMCommonDisp(hl, hl.design)
>>> Warning message:
>>> In estimateGLMCommonDisp.default(y = y$counts, design = design, :
>>> No residual df: setting dispersion to NA
>>>
>>> I tried to figure out what I have done wrong, unfortunately, I have no
>>> luck on that. Could you or the community kindly please light me for
>>> this matter?
>>>
>>> Thank you very much for your time.
>>>
>>>> sessionInfo()
>>>
>>> R version 2.15.1 (2012-06-22)
>>> Platform: i486-pc-linux-gnu (32-bit)
>>>
>>> locale:
>>> [1] LC_CTYPE=en_US.utf8 LC_NUMERIC=C
>>> [3] LC_TIME=en_US.utf8 LC_COLLATE=en_US.utf8
>>> [5] LC_MONETARY=en_US.utf8 LC_MESSAGES=en_US.utf8
>>> [7] LC_PAPER=C LC_NAME=C
>>> [9] LC_ADDRESS=C LC_TELEPHONE=C
>>> [11] LC_MEASUREMENT=en_US.utf8 LC_IDENTIFICATION=C
>>>
>>> attached base packages:
>>> [1] stats graphics grDevices utils datasets methods base
>>>
>>> other attached packages:
>>> [1] edgeR_2.6.10 limma_3.12.1
>>>
>>> loaded via a namespace (and not attached):
>>> [1] tcltk_2.15.1 tools_2.15.1
>>>
>>> Best regards,
>>> KJ Lim
>>>
>>>
>>>
>>> On 31 July 2012 02:24, Gordon K Smyth <smyth at wehi.edu.au> wrote:
>>>>
>>>> Dear KH Kim,
>>>>
>>>> No, you have not understood me correctly. I did not suggest that you
>>>> change
>>>> from edgeR to limma. I suggested that you read the limma documentation
>>>> because the design matrix is the same for edgeR as it is for limma, so
>>>> the
>>>> limma documentation would help you create the design matrix for your
>>>> edgeR
>>>> analysis.
>>>>
>>>> I thought from your last email that you had already done this and that
>>>> you
>>>> had completed the edgeR analysis satisfactorily.
>>>>
>>>> I wrote more documentation for the edgeR User's Guide a couple of days
>>>> ago,
>>>> trying to give advice for experiments such as yours. You might find that
>>>> Section 3.3 of
>>>>
>>>>
>>>> http://bioconductor.org/packages/2.11/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf
>>>>
>>>> gives more explanation.
>>>>
>>>> Best wishes
>>>> Gordon
>>>>
>>>> ---------------------------------------------
>>>> Professor Gordon K Smyth,
>>>> Bioinformatics Division,
>>>> Walter and Eliza Hall Institute of Medical Research,
>>>> 1G Royal Parade, Parkville, Vic 3052, Australia.
>>>> Tel: (03) 9345 2326, Fax (03) 9347 0852,
>>>> http://www.statsci.org/smyth
>>>>
>>>>
>>>> On Mon, 30 Jul 2012, KJ Lim wrote:
>>>>
>>>>> Dear Prof Gordon,
>>>>>
>>>>> Good day.
>>>>>
>>>>> I have read the section 8.5 of the Limma manual as you suggested in
>>>>> previous emails. Thanks.
>>>>>
>>>>> If I understand you correctly, you would suggest me to carry out my DE
>>>>> analysis with limma package if I would like to learn the which genes
>>>>> are express in treeHS compare to treeLS (vice versa) at i.e. 24H.
>>>>>
>>>>> May I ask how can I generate the EList, "eset", in order to fit in the
>>>>> linear model as mentioned in the limma manual
>>>>>
>>>>> fit <- lmFit(eset, design)
>>>>> fit <- eBayes(fit)
>>>>>
>>>>> Please correct me if I'm wrong.
>>>>>
>>>>> Thank you very much for your time and help.
>>>>>
>>>>> Best regards,
>>>>> KJ Lim
>>>>>
>>>>>
>>>>>
>>>>> On 27 July 2012 02:31, Gordon K Smyth <smyth at wehi.edu.au> wrote:
>>>>>>
>>>>>>
>>>>>> Dear KJ Lim,
>>>>>>
>>>>>> Thanks for the rephrasing, which is clearer. I would have liked you to
>>>>>> mention however whether you read the documentation that I refered you
>>>>>> do.
>>>>>>
>>>>>>
>>>>>> On Thu, 26 Jul 2012, KJ Lim wrote:
>>>>>>
>>>>>>> Dear Prof Gordon,
>>>>>>>
>>>>>>> Good day. Thanks for your prompt replied.
>>>>>>>
>>>>>>> Please allow me to rephrase my previous question.
>>>>>>>
>>>>>>> This model, ~tree+treat, is assumed effect of the time of treatment
>>>>>>> will be the same irrespective of genotype.
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Yes, this model makes that assumption.
>>>>>>
>>>>>>
>>>>>>> Thus, test for the coef=3 will gives me the differential expression at
>>>>>>> 96H
>>>>>>> irrespective of genotype.
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Actually coef=3 refers to 24H, according to the design matrix in your
>>>>>> original email. A test for coef=3 would test for DE at 24H vs 0H,
>>>>>> irrespective of genotype. But only if the assumption mentioned above
>>>>>> is
>>>>>> true, which is unlikely given the rest of your email.
>>>>>>
>>>>>>
>>>>>>> I would like to learn the differential expression of treeHS vs treeLS
>>>>>>> at specific time points i.e. 24H or if possible across the whole time
>>>>>>> points. Should I fit my model as
>>>>>>>
>>>>>>> model A: ~tree*treat OR model B: ~tree+tree:treat ?
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> These models are equivalent, so it is just a matter of convenience
>>>>>> which
>>>>>> one
>>>>>> you use, as I tried to explain in the limma documentation I refered you
>>>>>> to.
>>>>>>
>>>>>>
>>>>>>> The design matrix columns of model B give:
>>>>>>> "(Intercept)" "treeLS" "treeHS:treat03H" "treeLS:treat03H"
>>>>>>> "treeHS:treat24H" "treeLS:treat24H" "treeHS:treat96H"
>>>>>>> "treeLS:treat96H"
>>>>>>>
>>>>>>> Am I doing right if I fit the model B and test for the coef=3:4 to
>>>>>>> learn the differential expression of treeHS vs treeLS at specific time
>>>>>>> points i.e. 03H? Does this test could tells me which set of genes
>>>>>>> up/down-regulated in treeLS or treeHS?
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Yes. Coef 3 tests for a 3H effect in HS. Coef 4 tests for a 3H in LS.
>>>>>> So
>>>>>> testing for both coefficients coef=3:4 tests for a 3H effect in either
>>>>>> treeLS or treeHS.
>>>>>>
>>>>>> Best wishes
>>>>>> Gordon
>>>>>>
>>>>>>
>>>>>>> I'm not good in statistic and I'm learning, kindly please correct me
>>>>>>> if I'm wrong.
>>>>>>>
>>>>>>> Thank you very much for your time and suggestion.
>>>>>>>
>>>>>>> Best regards,
>>>>>>> KJ Lim
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>>
>>>>>>> On 26 July 2012 03:39, Gordon K Smyth <smyth at wehi.edu.au> wrote:
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>> Dear KJ Lim,
>>>>>>>>
>>>>>>>> I don't quite understand your question, because you seem to be asking
>>>>>>>> for
>>>>>>>> something that isn't a test for differential expression, which is
>>>>>>>> what
>>>>>>>> edgeR
>>>>>>>> does. You question "I would like to learn the genes are express in
>>>>>>>> treeHS
>>>>>>>> but not treeLS and vice versa" seems to be about absolute expression
>>>>>>>> levels.
>>>>>>>> edgeR doesn't test for genes that are not expressed in particular
>>>>>>>> conditions.
>>>>>>>>
>>>>>>>> I'll refer you to the limma section on interaction models in case
>>>>>>>> that
>>>>>>>> helps, see Section 8.5 of:
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>>
>>>>>>>> http://bioconductor.org/packages/2.11/bioc/vignettes/limma/inst/doc/usersguide.pdf
>>>>>>>>
>>>>>>>> Setting up a design matrix is the same for edgeR as it is for limma.
>>>>>>>>
>>>>>>>> Best wishes
>>>>>>>> Gordon
>>>>>>>>
>>>>>>>>> Date: Tue, 24 Jul 2012 17:12:00 +0300
>>>>>>>>> From: KJ Lim <jinkeanlim at gmail.com>
>>>>>>>>> To: Bioconductor mailing list <bioconductor at r-project.org>
>>>>>>>>> Subject: [BioC] edgeR: design matrix for different condition
>>>>>>>>>
>>>>>>>>> Dear the edgeR community,
>>>>>>>>>
>>>>>>>>> Good day.
>>>>>>>>>
>>>>>>>>> I'm analyzing my RNA-Seq experiment with edgeR. My study has 2
>>>>>>>>> different genotypes (treeHS, treeLS) and time of treatment (0H, 3H,
>>>>>>>>> 24H, 96H).
>>>>>>>>>
>>>>>>>>> I first assumed that the treatment effect will be the same for each
>>>>>>>>> genotype and I have the design matrix as:
>>>>>>>>>
>>>>>>>>> design <- model.matrix(~tree+treat)
>>>>>>>>>
>>>>>>>>>> design
>>>>>>>>>
>>>>>>>>>
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> (Intercept) treat03H treat24H treat96H treeLS
>>>>>>>>> 1 1 0 0 0 0
>>>>>>>>> 2 1 0 0 0 0
>>>>>>>>> 3 1 1 0 0 0
>>>>>>>>> 4 1 1 0 0 0
>>>>>>>>> 5 1 0 1 0 0
>>>>>>>>> 6 1 0 1 0 0
>>>>>>>>> 7 1 0 0 1 0
>>>>>>>>> 8 1 0 0 1 0
>>>>>>>>> 9 1 0 0 0 1
>>>>>>>>> 10 1 0 0 0 1
>>>>>>>>> 11 1 1 0 0 1
>>>>>>>>> 12 1 1 0 0 1
>>>>>>>>> 13 1 0 1 0 1
>>>>>>>>> 14 1 0 1 0 1
>>>>>>>>> 15 1 0 0 1 1
>>>>>>>>> 16 1 0 0 1 1
>>>>>>>>>
>>>>>>>>> I used coef=4 to test for the differential expressions between
>>>>>>>>> treeHS
>>>>>>>>> and treeLS within the time of treatment, coef=3 to learn the
>>>>>>>>> differential expressions in 2 genotypes at time of treatment 96H.
>>>>>>>>>
>>>>>>>>> ** I would like to learn the genes are express in treeHS but not
>>>>>>>>> treeLS and vice versa at certain time of treatment let's say 24H or
>>>>>>>>> across the whole time of treatment, should I have the design matrix
>>>>>>>>> as
>>>>>>>>> below or more steps I need to do?
>>>>>>>>>
>>>>>>>>> design <- model.matrix(~tree*treat)
>>>>>>>>>
>>>>>>>>> Kindly please light me on this. I appreciate very much for your help
>>>>>>>>> and
>>>>>>>>> time.
>>>>>>>>>
>>>>>>>>> Have a nice day.
>>>>>>>>>
>>>>>>>>> Best regards,
>>>>>>>>> KJ Lim
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