Question

Error in designing DESeq2 time pointed experiments

0

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AZ ▴ 30

@fereshteh-15803

Last seen 12 months ago

United Kingdom

I have a treatment and control in two time points like this

 > design
                                 X condition time
    1       CTRL_24_hrs_replicate1   control   24
    2       CTRL_24_hrs_replicate2   control   24
    3       CTRL_24_hrs_replicate3   control   24
    4  treatment_24_hrs_replicate1        t   24
    5  treatment_24_hrs_replicate2        t   24
    6  treatment_24_hrs_replicate3        t   24
    7       CTRL_48_hrs_replicate1   control   48
    8       CTRL_48_hrs_replicate2   control   48
    9       CTRL_48_hrs_replicate3   control   48
    10 treatment_48_hrs_replicate1        t   48
    11 treatment_48_hrs_replicate2        t   48
    12 treatment_48_hrs_replicate3        t   48
    >

I want to test between treatment and control considering time point 24 hours to 48 hours

I have done like this

dds <- DESeqDataSetFromMatrix(countData=a,colData=design, design=~time + condition + time:condition)

But at this part I get error, although I am trying different things

> ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + time:condition)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
Error in nbinomLRT(object, full = full, reduced = reduced, quiet = quiet,  : 
  less than one degree of freedom, perhaps full and reduced models are not in the correct order

or

> ddsTC <- DESeq(dds, test="LRT", reduced = ~time:condition)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
Error in nbinomLRT(object, full = full, reduced = reduced, quiet = quiet,  : 
  less than one degree of freedom, perhaps full and reduced models are not in the correct order
>

I tried these with no error although I am not certain if this makes sense at all

> ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + condition)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
> 

> ddsTC <- DESeq(dds, test="LRT", reduced = ~ time)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing

Al I want is getting the difference of treatment versus control but considering time goes from 24 hours to 48 hours

Thanks for any help

LTR DESeq2 • 797 views

ADD COMMENT • link 2.5 years ago AZ ▴ 30

score 1 · Answer 1 · 2021-10-26

1

Entering edit mode

Michael Love 41k

@mikelove

Last seen 12 hours ago

United States

The last design is the one we use at the end of the workflow (look up the rnaseqGene workflow)

I strongly recommend working with a statistician who can explain what these designs do rather than just seeing which run without error.

ADD COMMENT • link 2.5 years ago Michael Love 41k

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Thanks a lot

Having > ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + condition) I will have

> resultsNames(ddsTC)
[1] "Intercept"               "time_48_vs_24"           "condition_IT_vs_control"
[4] "time48.conditionIT"     
>

I asked somebody and he told me condition_IT_vs_control contrast will give me

the difference of treatment versus control but considering time goes from 24 hours to 48 hours

, since time is compensated for by fitting all coefficients at the same time

Now my problem is if I could get this by this design or not ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + condition)

ADD REPLY • link 2.5 years ago AZ ▴ 30

0

Entering edit mode

Please work with someone familiar with linear models in R to help design your analysis and interpret your results.

ADD REPLY • link 2.5 years ago Michael Love 41k

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Thank you so much

I noticed the results of ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + condition) and ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + condition) is exactly the same

Sorry, how I can get output by ddsTC <- DESeq(dds, test="LRT", reduced = ~ time + time:condition). with no error

I guess the right error is this one

ADD REPLY • link 2.5 years ago AZ ▴ 30