Question: Conversion from counts to FPKM
0
5 months ago by
chiara.facciotto0 wrote:

Hi,

I am trying to use DESeq2 to convert raw counts to fpkm, so I can compare gene aboundance across genes and not only across samples. I have a couple of questions on how to do so:

1. Should I first normalize the counts and transform them with vst and then use the fpkm() function, or should I simply input the raw counts and the fkpm() function will then take care of normalization as well?
2. How do I make sure that the genes in my GRanges object containing the gene lengths match the genes in the dss object?

Thank you!

modified 8 weeks ago by Ahmed Alhendi0 • written 5 months ago by chiara.facciotto0
Answer: Conversion from counts to FPKM
0
5 months ago by
Michael Love23k
United States
Michael Love23k wrote:

You should input the raw counts and then use fpkm() to generate FPKM values. The default is to use a robust estimate of library size (median ratio normalization) in place of the total count which is a sub-optimal estimator. Note that FPKM are not variance stabilized.

It is up to you to provide exonic basepair lengths, we don't have any code for that. An easier approach is to use a pipeline, such as Salmon followed by tximport, which keeps all the information together for you (and provides a much more accurate estimate of the length of the gene, using the average transcript length, as opposed to the sum of the exonic basepairs).

Chiara, as per Michael, FPKM units are not variance stabilised, and neither are they comparable across samples. There is no cross-sample normalisation employed when deriving FPKM expression units.

Answer: Conversion from counts to FPKM
0
8 weeks ago by
University of Leicester, UK
Ahmed Alhendi0 wrote:

Try countToFPKM package. This package provides an easy to use function to convert the read count matrix into FPKM matrix. Implements the following equation:

$enter image description here$

The fpkm() function requires three inputs to return FPKM as numeric matrix normalized by library size and feature length:

• counts A numeric matrix of raw feature counts.
• featureLength A numeric vector with feature lengths that can be obtained using
biomaRt.
• meanFragmentLength A numeric vector with mean fragment lengths,
which can be calculate with
Picard using CollectInsertSizeMetrics.