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Question: time-series, no replicates, no control groupe
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gravatar for bahlv
3 months ago by
bahlv0
bahlv0 wrote:

Hi,

I'm working with time series RNA-seq data, containig 6 time points. There are no replicates and there is no control groupe. I'm interested to find differential expressed genes between the first timepoint (1/reference) compared to all the other timepoints. The time points can be grouped in 3 groups like this: condition A: 1,2  condition B: 3,4,5  condition C: 6 (but they are not replicates).

I first tried to do a multifactor design with the design (in order to do a likelihood ratio test later):

colData=data.frame(row.names=colnames(ei2),
                   timepoint=c("1", "2", "3", "4", "5", "6"),
                   condition=c("A", "A", "B", "B","B", "C"))

design(ddsMF)=formula(~condition+timepoint)

ddsMF=DESeq(ddsMF)

but it gives back the error:

Error in checkFullRank(modelMatrix) : 
  the model matrix is not full rank, so the model cannot be fit as specified.
  One or more variables or interaction terms in the design formula are linear
  combinations of the others and must be removed.

I don't know how to fix the problem with the model matrix neither if the approach is right. Are there any suggestions on how to solve this problem or antoher way to find differential expressed genes?

Thanks in advance!

   

 

   
     
     
     
     
ADD COMMENTlink modified 3 months ago by Michael Love19k • written 3 months ago by bahlv0
0
gravatar for Michael Love
3 months ago by
Michael Love19k
United States
Michael Love19k wrote:

DESeq2 requires replicates and so you cannot compare each time point to the first one, but you can code time as a numeric and use a smooth function to model the counts. Eg a design of f(time) using a smooth function ‘f’ such as splines or polynomials, with a likelihood ratio test comparing to a reduced design of ~1. You should contact a local statistician to assist with the modeling choices if you have not used splines or polynomial functions in linear models in R before.

ADD COMMENTlink written 3 months ago by Michael Love19k

Thanks for the quick reply!

Since i'm completly new to DGE analysis, could you give me another advise on how to analyse this dataset? Probably another tool which fits better?

I also thought about comparing each timepoint to the refernce with the help of contrasts (which I know is not the best, since the timepoints are treated as replicates for dispersion estimates)

ADD REPLYlink written 3 months ago by bahlv0

Above is my best advice for how to analyze the dataset and doesn't really have to do with any specific packages.

ADD REPLYlink written 3 months ago by Michael Love19k
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