Hi, Mike:

 

Thanks for the help. Now we have updated the R and bioconductor version as well as the DESeq2. Here is sessionInfo() 

R version 3.0.3 (2014-03-06)
Platform: x86_64-unknown-linux-gnu (64-bit)

[1] DESeq2_1.2.10 

 

All the function calls now seem working, But I still got some issues for the contrasts I desired;

 

> dds <- DESeqDataSetFromMatrix(countData = countD,colData = colD,design = ~Type + RasType + Type:RasType);
Usage note: the following factors have 3 or more levels:

RasType

For DESeq2 versions < 1.3, if you plan on extracting results for
these factors, we recommend using betaPrior=FALSE as an argument
when calling DESeq().
...

 

> colData(dds)$RasType <- factor(colData(dds)$RasType,levels=c("RasOnly","RasP","RasP1Hit","RasPNot"));

> colData(dds)$Type <- factor(colData(dds)$Type,levels=c("Normal","Tumor"))
> dds<-DESeq(dds, betaPrior=FALSE);
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing

> show(resultsNames(dds));
[1] "Intercept"                   "Type_Tumor_vs_Normal"
[3] "RasType_RasP_vs_RasOnly"     "RasType_RasP1Hit_vs_RasOnly"
[5] "RasType_RasPNot_vs_RasOnly"  "TypeTumor.RasTypeRasP"
[7] "TypeTumor.RasTypeRasP1Hit"   "TypeTumor.RasTypeRasPNot"


> res_RasP_vs_RasOnly <- results(dds,"RasType_RasP_vs_RasOnly")
> res_RasP_vs_RasOnly <- results(dds,name="RasType_RasP_vs_RasOnly")
> res_Tumor_vs_Normal<-results(dds,contrast=c("Type","Tumor","Normal"))
> res_RasP_vs_RasOnly <- results(dds,contrast=c("RasType","RasP","RasOnly"))
> res_Tumor.RasP_vs_Tumor.RasPNot<-results(dds,contrast=c(0,0,0,0,0,1,0,-1))

I can get above contrast results working and got the DEGs lists. However, although I can get contrast result Tumor.RasP_vs_Tumor.RasPNot using contrast=c(0,0,0,0,0,1,0,-1) as guide indicated, some of desired contrasts I am not sure how to get:

I have 4 levels of RasType:  "RasOnly","RasP","RasP1Hit","RasPNot", it seems that RasOnly might be in intercept, since I did not see it in show(resultsNames(dds)). And I am interested in contrast: Tumor.RasOnly_Tumor.RasPNot, how do I get the results for this contrast? 

my best guess is if TypeTumor.RasTypeRasOnly is at intercept, I can potentially get using below:

Res_Tumor.RasOnly_vs_Tumor.RasPNot <-results(dds, contrast=c(1,0,0,0,0,0,0,-1).  

 

However, I like to get the results of contrast: RasOnly.Normal_vs_RasPNot.Normal just to check normal contrast as background, then I am stuck, since no TypeNormal.RasTypeRasP contrast terms etc shown up in show(resultsNames(dds)).

 

Any advice here? 

 

Thanks so much!

Best

Ming

 

 

 

  

 

  

 


 

 

 

 

 



From: michaelisaiahlove@gmail.com
Date: Sun, 9 Mar 2014 13:35:34 -0400
Subject: Re: Questions about multi-factor contrast setting in DESeq2
To: yi02@hotmail.com
CC: bioconductor@r-project.org



hi Ming,


Are you using the latest release of DESeq2, version 1.2.x?  The contrast functionality was implemented in this release.


You can check the help for ?results to debug, i.e. to see if there is a 'contrast' argument in your installed version. You can check your versions with sessionInfo().  You can install latest versions with biocLite("DESeq2"), but you might need to upgrade to the latest release version of R, see the installation help on the Bioc website.


best


Mike



On Sun, Mar 9, 2014 at 1:11 PM, Ming Yi <yi02@hotmail.com> wrote:



Hi, Mike:
 
Thx for your advice again and I did try what you suggested as below:
 
> dds <- DESeqDataSetFromMatrix(countData = countD,colData = colD,design = ~Type + RasType + Type:RasType); 

> dds <- DESeq(dds);
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting generalized linear model
105 rows did not converge in beta, labelled in mcols(object)$betaConv. Use larger maxit argument with nbinomWaldTest
> show(resultsNames(dds)); 

[1] "Intercept"                   "Type_Tumor_vs_Normal"
[3] "RasType_RasP_vs_RasOnly"     "RasType_RasP1Hit_vs_RasOnly"
[5] "RasType_RasPNot_vs_RasOnly"  "TypeTumor.RasTypeRasP"
[7] "TypeTumor.RasTypeRasP1Hit"   "TypeTumor.RasTypeRasPNot"

certainly we can use 
> res_RasP_vs_RasOnly <- results(dds,"RasType_RasP_vs_RasOnly")
> res_RasP_vs_RasOnly <- results(dds,name="RasType_RasP_vs_RasOnly")
> res_Tumor_vs_Normal<-results(dds,"Type_Tumor_vs_Normal")
> res_Tumor_vs_Normal<-results(dds,name="Type_Tumor_vs_Normal")
 
but I can not do the following with contrast as suggested in section 3.2:
> res_Tumor_vs_Normal<-results(dds,contrast=c("Type","Tumor","Normal"))
Error in results(dds, contrast = c("Type", "Tumor", "Normal")) :
  unused argument (contrast = c("Type", "Tumor", "Normal"))
>  res_RasP_vs_RasOnly <- results(dds,contrast=c("RasType","RasP","RasOnly"))
Error in results(dds, contrast = c("RasType", "RasP", "RasOnly")) :
  unused argument (contrast = c("RasType", "RasP", "RasOnly"))
 
also I can not get contrast like Tumor.RasP_Tumor.RasPNot:
> res_Tumor.RasP_Tumor.RasPNot<-results(dds,contrast=c(0,0,0,0,0,1,0,-1))
Error in results(dds, contrast = c(0, 0, 0, 0, 0, 1, 0, -1)) :
  unused argument (contrast = c(0, 0, 0, 0, 0, 1, 0, -1))
 
it seems the interaction terms in the design (design = ~Type + RasType + Type:RasType) changed the behavior of results()?
 
Any idea or advice?
Thanks again for your time and help!
 
Ming
 



From: michaelisaiahlove@gmail.com
Date: Fri, 7 Mar 2014 19:12:23 -0500 


Subject: Re: Questions about multi-factor contrast setting in DESeq2
To: yi02@hotmail.com
CC: bioconductor@r-project.org



hi Ming,


To follow up on the question about contrasts, the way to perform comparisons like "RasOnly.Tumor vs RasOnly.Normal", would be a design:


Type + RasType + Type:RasType 


where:


results(dds, contrast=c("Type","Tumor","Normal"))


tests for the general effect,


and then the results for the interactions -- which are present in resultsNames(dds) and can be extracted using the 'name' argument to results() -- tests for an effect in a specific RasType which is different than the general effect.


Mike



On Fri, Mar 7, 2014 at 5:31 PM, Michael Love <michaelisaiahlove@gmail.com> wrote:


Hi Ming,
Exploratory data analysis might be a more fruitful approach here rather than brute force combinatorics and testing.
Copying from Wolfgang's recommendation in a similar situation:
"my advice here would be to put less emphasis on the testing and move straight to clustering, using one of the transformations described in the DESeq2 vignette to bring the data to a 'well-behaved' (log-like) scale."
"To filter out the genes that vary not much, use the range (max-min) or IQR and a subjective cutoff (e.g. retain the top 20% of genes), then use standard clustering functions (e.g. pam from the cluster package), and other exploratory data analyses (e.g. PCA) to see the types of behaviours."
You might also try constructing a heatmap, as shown in the vignette, using a subset of genes which vary the most, and then explore the grouping of samples in the hierarchical clustering on the columns. For ease of visualization, this subset should probably be in the 100s.
Mike


On Mar 7, 2014 2:59 PM, "Ming Yi" <yi02@hotmail.com> wrote:



Hi, Mike:
 
Thx for the info, indeed I did try the following before with  the following for the data in user guide:
> dds <- DESeqDataSetFromMatrix(countData = countData,colData = colData, design = ~ condition+type)
> dds <- DESeq(dds)
> resultsNames(dds)
[1] "Intercept"                      "condition_untreated_vs_treated"
[3] "type_single.read_vs_paired.end"
 
As you can see, the contrast I can get here is overall untreated_vs_treated and overall single.read_vs_paired.end, there is no subtype contrast such as treated.single.read vs treated.paired-end etc. 
 
I would love to discuss briefly what I need here. I have a dataset which has tumors and matched normal samples from many patients, and there are subtypes of the tumors, say RasOnly, RasP, RasPNot types of tumors, of course, corresponding matched would be also with subtypes of  RasOnly, RasP,RasP1Hit, RasPNot, and the metadata like this:

          Subject SampleName   Type  RasType          RasTum
T6745_01A 49_6745  T6745_01A  Tumor     RasP      RasP.Tumor
N6745_11A 49_6745  N6745_11A Normal     RasP     RasP.Normal
T6761_01A 49_6761  T6761_01A  Tumor  RasPNot   RasPNot.Tumor
N6761_11A 49_6761  N6761_11A Normal  RasPNot  RasPNot.Normal
T5930_01A 50_5930  T5930_01A  Tumor RasP1Hit  RasP1Hit.Tumor
N5930_11A 50_5930  N5930_11A Normal RasP1Hit RasP1Hit.Normal
T5932_01A 50_5932  T5932_01A  Tumor  RasOnly   RasOnly.Tumor
N5932_11A 50_5932  N5932_11A Normal  RasOnly  RasOnly.Normal
........
 
Here are the contrasts I am interested to get DEGs:
RasOnly.Tumor vs RasOnly.Normal 
RasP.Tumor vs RasP.Normal 
RasP.Tumor + RasP1Hit.Tumor vs RasP.Normal+RasP1Hit.Normal 
RasPNot.Tumor vs RasPNot.Normal
RasP1Hit.Tumor vs RasP1Hit.Normal
RasP.Tumor vs RasPNot.Tumor
RasP.Tumor+RasP1Hit.Tumor vs RasPNot.Tumor
RasOnly.Tumor vs RasPNot.Tumor
RasOnly.Normal vs RasPNot.Normal
RasP.Normal vs RasPNot.Normal
RasPRasP1Hit.Normal vs RasPNot.Normal,
 Tumor vs Normal
 
The last item Tumor vs Normal certainly can easily use design = ~ type to deal with. But many of the contrasts listed above not easy unless use the RasTum of the metadata shown above. I did try to use design=~Type+RasType 
here are the commands:
> dds <- DESeqDataSetFromMatrix(countData = countD,colData = colD,design = ~Type+RasType);
> show(resultsNames(dds))
character(0)
> dds <- DESeq(dds);
> resultsNames(dds)
[1] "Intercept"                   "Type_Tumor_vs_Normal"
[3] "RasType_RasP_vs_RasOnly"     "RasType_RasP1Hit_vs_RasOnly"
[5] "RasType_RasPNot_vs_RasOnly"

as you can see, I can only derive overall subtype contrasts from this way but not something like RasP.Tumor vs RasOnly.Tumor, the overall subtype contrasts for example, RasType_RasP_vs_RasOnly, consider both tumor and normal of RasP compared with those of RasOnly, which is certainly not what we want here. 
user guide section 3.2 did show 
resCtrst <- results(ddsCtrst, contrast=c("treatment","OHT","DPN"))
resCtrst <- results(ddsCtrst, contrast=c(0,0,0,0,-1,1))
 
So besides RasTum, if you have a better way using just design = ~Type+RasType, that would be great. 
I did try the following:
 
> dds <- DESeqDataSetFromMatrix(countData = countD,colData = colD,design = ~RasTum);
> dds <- DESeq(dds);
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting generalized linear model
172 rows did not converge in beta, labelled in mcols(object)$betaConv. Use larger maxit argument with nbinomWaldTest
> resultsNames(dds)
[1] "Intercept"
[2] "RasTum_RasOnly.Tumor_vs_RasOnly.Normal"
[3] "RasTum_RasP.Normal_vs_RasOnly.Normal"
[4] "RasTum_RasP.Tumor_vs_RasOnly.Normal"
[5] "RasTum_RasP1Hit.Normal_vs_RasOnly.Normal"
[6] "RasTum_RasP1Hit.Tumor_vs_RasOnly.Normal"
[7] "RasTum_RasPNot.Normal_vs_RasOnly.Normal"
[8] "RasTum_RasPNot.Tumor_vs_RasOnly.Normal"
 
I did get many contrasts as I desired, but the contrasts RasTum_RasP.Tumor_vs_RasOnly.Normal does not make sense here to me, but it shown up there in the resultsNames(dds) . 
 
based on section 3.2, I seem be able to derived more from the above contrasts:
say: for contrast RasP.Tumor vs RasP.Normal, I can do:
resCtrst<-result(dds, contrast=c(0,0,-1,1,0,0,0,0);
 
But is there any better way to do the above contrasts listed above that I desire?
 
Thx again for your advice!
best
 
Ming 
 
 
 
 
 
 
  
 



From: michaelisaiahlove@gmail.com
Date: Fri, 7 Mar 2014 13:23:56 -0500
Subject: Re: Questions about multi-factor contrast setting in DESeq2
To: yi02@hotmail.com
CC: bioconductor@r-project.org



hi Ming,


I'm confused why you are not following the instructions in the vignette section 1.5 Multifactor designs?  You should not and we do not recommend pasting together columns like this, nor inserting + 0 into the design. Please have a look at what we do recommend in this section. 


Extracting contrasts is covered in vignette section 3.2 Contrasts. First take a look at the entire vignette, as we've spent a lot of time writing the documentation to try to answer user questions.


we are happy to discuss the best approach for your experiment, but first we need to hear more about your aims and experiment e.g. what hypotheses you wish to test, what kind of genes you are looking to find. It's hard for us to reverse engineer a recommendation rather than to go at it from basic aims.


Mike



On Fri, Mar 7, 2014 at 12:52 PM, Ming Yi <yi02@hotmail.com> wrote:




Hi, Mike and All:
 
I am testing DESeq2 for multi-factor contrast setting for my own data with more complex meta data but currently use the simpler dataset from the user guide for testing purpose, and run into some issues that need your input and advice.  Here are the commands (only show some more relevant outputs):
 
library("DESeq2")
library("Biobase")
library("pasilla")
data("pasillaGenes")
countData <- counts(pasillaGenes)
colData <- pData(pasillaGenes)[,c("condition","type")]
colData<-data.frame(colData,paste(colData$condition,colData$type,sep="."))
colnames(colData)[3]<-"condition_type";
> dds <- DESeqDataSetFromMatrix(countData = countData,colData = colData, design = ~ condition_type)
> colData(dds)
DataFrame with 7 rows and 4 columns
             condition        type        condition_type sizeFactor
              <factor>    <factor>              <factor>  <numeric>
treated1fb     treated single-read   treated.single-read  1.5116926
treated2fb     treated  paired-end    treated.paired-end  0.7843521
treated3fb     treated  paired-end    treated.paired-end  0.8958321
untreated1fb untreated single-read untreated.single-read  1.0499961
untreated2fb untreated single-read untreated.single-read  1.6585559
untreated3fb untreated  paired-end  untreated.paired-end  0.7117763
untreated4fb untreated  paired-end  untreated.paired-end  0.783745
>dds <- DESeq(dds)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting generalized linear model
> resultsNames(dds)
[1] "Intercept"
[2] "condition_type_treated.single.read_vs_treated.paired.end"
[3] "condition_type_untreated.paired.end_vs_treated.paired.end"
[4] "condition_type_untreated.single.read_vs_treated.paired.end"
> colData(dds)
DataFrame with 7 rows and 4 columns
             condition        type        condition_type sizeFactor
              <factor>    <factor>              <factor>  <numeric>
treated1fb     treated single-read   treated.single-read  1.5116926
treated2fb     treated  paired-end    treated.paired-end  0.7843521
treated3fb     treated  paired-end    treated.paired-end  0.8958321
untreated1fb untreated single-read untreated.single-read  1.0499961
untreated2fb untreated single-read untreated.single-read  1.6585559
untreated3fb untreated  paired-end  untreated.paired-end  0.7117763
untreated4fb untreated  paired-end  untreated.paired-end  0.7837458

Then I tried  a slight diiffermry setting of the design:
> dds <- DESeqDataSetFromMatrix(countData = countData,colData = colData, design = ~0+ condition_type)
> dds <- DESeq(dds)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting generalized linear model
580 rows did not converge in beta, labelled in mcols(object)$betaConv. Use larger maxit argument with nbinomWaldTest
> resultsNames(dds)
[1] "condition_typetreated.paired.end"
[2] "condition_type_treated.single.read_vs_treated.paired.end"
[3] "condition_type_untreated.paired.end_vs_treated.paired.end"
[4] "condition_type_untreated.single.read_vs_treated.paired.end"
> colData(dds)
DataFrame with 7 rows and 4 columns
             condition        type        condition_type sizeFactor
              <factor>    <factor>              <factor>  <numeric>
treated1fb     treated single-read   treated.single-read  1.5116926
treated2fb     treated  paired-end    treated.paired-end  0.7843521
treated3fb     treated  paired-end    treated.paired-end  0.8958321
untreated1fb untreated single-read untreated.single-read  1.0499961
untreated2fb untreated single-read untreated.single-read  1.6585559
untreated3fb untreated  paired-end  untreated.paired-end  0.7117763
untreated4fb untreated  paired-end  untreated.paired-end  0.7837458

Then supposedly, I can use results(dds, "condition_type_treated.single.read_vs_treated.paired.end") to get DEGs for each contrast shown in resultsNames(dds).
 
here are my questions:
1. I used design = ~0+ condition_type instead of design = ~ condition_type in 2nd case, try to skip the intercept so that I can easily get all possible contrasts,  but seem not working the way I want.  
2. I tried to get all possible contrasts:  but besides the contrasts shown in resultsNames(dds) in both cases, the contrasts like  untreated.single.read vs treated.single.read, untreated.paired.end vs untreated.single.read not even exists in the resultsNames(dds). also I like the contrast generally like: treated (including both treated.single.read and treated.paired-end) vs untreated (including both untreated.single-read and untreated  paired-end). I know for this case, we can just to design = ~condition, but I wish to do this in the same roof of one single design model although I can do a separate design. In limma and edgeR, there is a function like: con.matrix<-makeContrasts() where one can set up any contrasts under the design at will. Is there anythign like that in DESeq2? I understand we can do  design(dds) <- formula(~ condition_type), but no contrast setting can be made at will. Anything in DESeq2 can get around that?
3. Also for simple contrast, I understand one can use relevel(colData(dds)$condition,"control") kind of command to set base level, but for multiple-factors contrasts as I am after, I almost need some kind of makeContrasts() mechanism to set up contrasts at will or have to do that individually one by one, which obvioulsy would be tedious and also these contrasts won't be in a single model roof. Anything can get around like that as well? if question 2 is addressed, this one shall be no problem.
 
Thanks in advance for your help! Appreciated very much!
 
Best
 
Ming 
ATRF/NCI-Frederick,
Maryland, USA.
 
    
 


 



 		 	   		  
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