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Question: Matrix is not full rank with Multifactorial Experiment and Time course
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gravatar for irving.jgalo23
12 days ago by
irving.jgalo230 wrote:

Hi

I have a multifactorial experiment with three factors: two levels of light treatment( high and low), two levels for tissues (cotyledon and hypocotyl), and finally I have a time course with four levels (15, 45, 90, 180 including the time 0). We have 57 libraries with replicates.

> coldata
            tissue   light time
000_A_C1 Cotyledon High_FR    0
000_A_C2 Cotyledon High_FR    0
000_A_H1 Hipocotyl High_FR    0
000_A_H2 Hipocotyl High_FR    0
000_A_H3 Hipocotyl High_FR    0
015_A_C1 Cotyledon High_FR   15
015_A_C2 Cotyledon High_FR   15
015_A_H1 Hipocotyl High_FR   15
015_A_H2 Hipocotyl High_FR   15
015_A_H3 Hipocotyl High_FR   15
015_A_H4 Hipocotyl High_FR   15
015_B_C1 Cotyledon  Low_FR   15
015_B_C2 Cotyledon  Low_FR   15
015_B_H1 Hipocotyl  Low_FR   15
015_B_H2 Hipocotyl  Low_FR   15
045_A_C1 Cotyledon High_FR   45
045_A_C2 Cotyledon High_FR   45
045_A_C3 Cotyledon High_FR   45
045_A_H1 Hipocotyl High_FR   45
045_A_H2 Hipocotyl High_FR   45
045_A_H3 Hipocotyl High_FR   45
045_A_H4 Hipocotyl High_FR   45
045_B_C1 Cotyledon  Low_FR   45
045_B_C2 Cotyledon  Low_FR   45
045_B_C3 Cotyledon  Low_FR   45
045_B_H1 Hipocotyl  Low_FR   45
045_B_H2 Hipocotyl  Low_FR   45
045_B_H3 Hipocotyl  Low_FR   45
045_B_H4 Hipocotyl  Low_FR   45
090_A_C1 Cotyledon High_FR   90
090_A_C2 Cotyledon High_FR   90
090_A_C3 Cotyledon High_FR   90
090_A_C4 Cotyledon High_FR   90
090_A_H1 Hipocotyl High_FR   90
090_A_H2 Hipocotyl High_FR   90
090_A_H3 Hipocotyl High_FR   90
090_A_H4 Hipocotyl High_FR   90
090_B_C1 Cotyledon  Low_FR   90
090_B_C2 Cotyledon  Low_FR   90
090_B_C3 Cotyledon  Low_FR   90
090_B_H1 Hipocotyl  Low_FR   90
090_B_H2 Hipocotyl  Low_FR   90
090_B_H3 Hipocotyl  Low_FR   90
090_B_H4 Hipocotyl  Low_FR   90
180_A_C1 Cotyledon High_FR  180
180_A_C2 Cotyledon High_FR  180
180_A_C3 Cotyledon High_FR  180
180_A_H1 Hipocotyl High_FR  180
180_A_H2 Hipocotyl High_FR  180
180_A_H3 Hipocotyl High_FR  180
180_B_C1 Cotyledon  Low_FR  180
180_B_C2 Cotyledon  Low_FR  180
180_B_C3 Cotyledon  Low_FR  180
180_B_C4 Cotyledon  Low_FR  180
180_B_H1 Hipocotyl  Low_FR  180
180_B_H2 Hipocotyl  Low_FR  180
180_B_H3 Hipocotyl  Low_FR  180

To start analyzing the data, I designed the following model

coldata

> dds <- DESeqDataSetFromMatrix(
  countData = countData,
  colData = coldata,
  design = ~ light + time + tissue + light:tissue)
> ds

Pre-filtering runs collapsed
dds <- ddsCollapsed[ rowSums(counts(ddsCollapsed)) > 50, ]
dds

I have been able to perform a contrast of the light treatment

##Deseq2
> dds <- DESeq(dds)
> resultsNames(dds)
#Contrast high light vs low light
> res_llow_vs_lhigh <- results(dds,contrast=c("light","Low_FR","High_FR"))
> res_llow_vs_lhigh <- res_llow_vs_lhigh[order(res_llow_vs_lhigh$padj),]  # Sorting

> head (res_llow_vs_lhigh)
log2 fold change (MLE): light Low_FR vs High_FR 
Wald test p-value: light Low FR vs High FR 
DataFrame with 6 rows and 6 columns
           baseMean log2FoldChange     lfcSE      stat       pvalue         padj
          <numeric>      <numeric> <numeric> <numeric>    <numeric>    <numeric>
AT1G02340 2083.4933       3.361691 0.1856905  18.10373 2.978046e-73 6.302439e-69
AT4G16780 2473.4689       4.248042 0.2401146  17.69173 4.856344e-70 5.138741e-66
AT1G04180  134.6161       6.273875 0.4672011  13.42864 4.108919e-41 2.898569e-37
AT3G21330  821.5631       5.059732 0.4170330  12.13269 7.088210e-34 3.750195e-30
AT1G21050  994.0203       1.477917 0.1245895  11.86229 1.858119e-32 6.593775e-29
AT2G46970  679.6482       6.868255 0.5790236  11.86179 1.869425e-32 6.593775e-29
 
 

But now Im interested to view the genes that are differentially expressed in the time course comparing between tissues, but when I modify the model design with the time interactions it throws me an error : "Model is not full rank".

> dds <- DESeqDataSetFromMatrix(
+   countData = countData,
+   colData = coldata,
+   design = ~ light + time + tissue + light:tissue + light:time)
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.

  Please read the vignette section 'Model matrix not full rank':

  vignette('DESeq2')

I read the vignette and I think that the possible reason is the first: one or more columns in my model matrix are linear combinations of other columns. Could you help me fix my data set to turn it into a model matrix full rank?

Another question is How can I analyze a time series experiment? I read the vignette from "airway", but I have doubts about how they set up the dataset in the package?

Regards

ADD COMMENTlink modified 12 days ago by Michael Love14k • written 12 days ago by irving.jgalo230
1
gravatar for Michael Love
12 days ago by
Michael Love14k
United States
Michael Love14k wrote:

First, note that when you include an interaction term for light:tissue, the above contrast is the effect of light treatment for the reference tissue. You can see the interactions diagram in the DESeq2 vignette, but I think you should also consider consulting a local statistician. I can't immediately parse from "differentially expressed in the time course comparing between tissues" exactly what the proper design should be. It involves a longer conversation about the desired contrasts, and how to take into account the light treatment.

The technical problem you encounter when adding light:time is that this would be giving you the differences in the high vs low light effect over time relative to the difference between high and low light at time=0. But you don't have low light samples at time=0. This is tricky to resolve because you have the other interaction with light in the model already.

ADD COMMENTlink written 12 days ago by Michael Love14k

Hi Michael, thanks for your answer. In this case, I want to compare the time 0 vs all the times (0 vs 15, 0 vs 45 ..... etc), to observe through time that genes change in contrast to time 0, both in cotyledons and in hypocotyls while you apply the light treatment. But I think that I have to consider the interactions.

What do you recommend me?

Regards

ADD REPLYlink written 12 days ago by irving.jgalo230

It’s a relatively complex design so I’d recommend meeting with a statistician so you get the design and contrasts that you want. There is nothing special about the way DESeq2 will generate coefficients from standard R formula for linear models.

ADD REPLYlink written 12 days ago by Michael Love14k
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