Question: calculate p-value/q-value use fpkm - RNA-Seq
gravatar for AnneC
15 months ago by
AnneC0 wrote:

Hello everyone!

We got an excel file containing gene and fpkm, how could I get significant DEG by using fpkm?

The data is from RNA-Seq. We have 3 replication for control and case.

deg q-value rna-seq fpkm pvalue • 1.3k views
ADD COMMENTlink modified 15 months ago by Kevin Blighe53k • written 15 months ago by AnneC0

Can't you get read counts instead of fpkms? Popular and reliable software for DEG analysis work with raw read counts.

ADD REPLYlink written 15 months ago by grant.hovhannisyan1.8k
gravatar for Kevin Blighe
15 months ago by
Kevin Blighe53k
Kevin Blighe53k wrote:

Regarding differential expression analysis and FPKM, please read these:

You should abandon RPKM / FPKM. They are not ideal where cross-sample differential expression analysis is your aim; indeed, they render samples incomparable via differential expression analysis:

Please read this: A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis

The Total Count and RPKM [FPKM] normalization methods, both of which are still widely in use, are ineffective and should be definitively abandoned in the context of differential analysis.

Also, by Harold Pimental: What the FPKM? A review of RNA-Seq expression units

The first thing one should remember is that without between sample normalization (a topic for a later post), NONE of these units are comparable across experiments. This is a result of RNA-Seq being a relative measurement, not an absolute one.


You should aim to obtain the raw counts for your dataset of interest and then reprocess these using a normalisation strategy that is more amenable to differential expression (like those implemented in DESeq2, EdgeR, and Limma in R Programming Language).

You will not find much advice for conducting statistical comparisons on FPKM data in Excel on this forum, but I could be proved incorrect on that.


ADD COMMENTlink modified 15 months ago • written 15 months ago by Kevin Blighe53k

Thanks very much for your advice, Kevin!

ADD REPLYlink written 15 months ago by AnneC0
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