Question: In-sample and across samples normalized expression
1
gravatar for Rajinder Gupta
5 weeks ago by
Netherlands
Rajinder Gupta10 wrote:

I want to get the expression data that is in-sample normalized like FPKM and also across samples normalized as obtained using DESeq2 or else.

What I am currently doing is that I first normalize the data across samples (using DESeq) and from the resultant expression I calculate the FPKM. Does it make sense or am I missing something here?

ADD COMMENTlink written 5 weeks ago by Rajinder Gupta10
1

The data have already been scaled for depth after the DESeq2 normalization, so FPKM is not suitable. Why do you want to do that?

ADD REPLYlink written 5 weeks ago by ATpoint24k

With DESeq the data is normalized across samples but I also want to compare different transcripts within the same sample which is not possible with DESeq normalization because of the different sizes of the transcripts. With FPKM this can be acheived but then how do I address the differences in sequencing depth across different samples.

ADD REPLYlink written 5 weeks ago by Rajinder Gupta10

I think as these are two different analysis goals, use TPM to compare your transcripts within the sample and use DESeq2 to compare samples. They are not easily interchangable. Is there an argument from your side not to do both separately?

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by ATpoint24k

Unfortunately yes. I am developing a pipeline for analyses in which I have to use the results from within sample comparison for the across sample comparison

ADD REPLYlink written 5 weeks ago by Rajinder Gupta10

That is not a good idea. Please use google and the search function on why FPKM/TPM perform poorly for inter-sample comparison. There is a lot of literature out there on this plus the authors of the established differential analysis tools recommend explicitely against doing that. If you browse Bioconductor support page a bit you'll see why.

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by ATpoint24k

I understand the limitation of use of FPKM for inter-sample comparison i.e. I am thinking of calculating the FPKM from the normalized read counts. What I am proposing is that the samples are first normalized using DESeq, edgeR or else and then from these normalized counts FPKM is calculated. So this is not the direct FPKM but an intersample normalized FPKM

ADD REPLYlink modified 5 weeks ago • written 5 weeks ago by Rajinder Gupta10
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