Question: Different PCA plots using rlog and vsd on the same data set
0
gravatar for lirongrossmann
2.7 years ago by
lirongrossmann20 wrote:

Hi All,

I have been using Deseq2 to analyze a dataset I have and ran into a problem I am not sure how to solve.

I have been using the following code to run deseq2 on my dataset:

dds <-DESeqDataSetFromMatrix(countData = ep,colData = cp,design = ~Risk)
dds <- estimateSizeFactors(dds)
rld <- rlog(dds)
plotPCA(rld, intgroup="Risk")
vsd <- varianceStabilizingTransformation(dds)
plotPCA(vsd, intgroup="Risk")

The two PCA plots I got look completely different, so I am not sure which transformation I should rely on for further analysis.

Any help?

rna-seq pca deseq2 • 2.1k views
ADD COMMENTlink modified 2.7 years ago by geek_y11k • written 2.7 years ago by lirongrossmann20
1

Just a comment that may be of interest:

DESeq2's PCA function automatically removes a proportion of variables of low variance before printing the plot. As you're using a variance-stabilised transformation versus a regularised log transformation, I would fully expect the PCA plots to look different based on this behaviour of DESeq2.

If you try the code that I posted in your other thread on both the rld and vst counts, then I think that you will see more similar PCA bi-plots, as, with my code, no variables are removed prior to generating the plots.

ADD REPLYlink modified 2.7 years ago • written 2.7 years ago by Kevin Blighe60k

Thank you! I have been using variance-stabilised transformation mainly because rlog runs very slowly. In your experience, does rlog give better results?

ADD REPLYlink written 2.7 years ago by lirongrossmann20

I prefer rlog counts purely because the data distribution looks better, however, as you mentioned, it can be very very slow! There is nothing wrong with using variance-stabilised counts.

In DESeq2, you have to remember, too, that the statistical tests are performed on just the normalised counts, i.e., before any rlog or vst.

ADD REPLYlink written 2.7 years ago by Kevin Blighe60k

You should post the PCA Plot. You can anonymise the sample labels if you don't wish to show the sample labels.

ADD REPLYlink written 2.7 years ago by geek_y11k

I would love to! Is there a way to upload the plots?

ADD REPLYlink written 2.7 years ago by lirongrossmann20

You'll need to upload them elsewhere and then link to them.

ADD REPLYlink written 2.7 years ago by Devon Ryan95k

Please add links when you cross post to multiple forum sites.

ADD REPLYlink written 2.7 years ago by Michael Love2.0k
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