Question: TMM - Normalized FPKM values for RNASeq data ?
4
gravatar for gangireddy
2.2 years ago by
gangireddy160
gangireddy160 wrote:

Hi People,

I want to generate TMM- Normalized fpkm values RNASeq data 5 time point data for male and female. I am very much confused how to generate these values.

1) Is it applying TMM to fpkm or apply TMM to raw counts and log transform the values to fpkm ?

2) Is it acceptable to use TMM- Normalized fpkm or should people use TMM- normalized raw count directly ?

and finally

3) TMM- normalized fpkm does it mean it normalized twice and is it necessary.

Can anyone provide the step wise commands in R to generate TMM- normalized fpkm matrix

Thanks in advance

rna-seq next-gen R • 2.2k views
ADD COMMENTlink modified 2.2 years ago by EVR470 • written 2.2 years ago by gangireddy160
1

What would you like to do with these TMM-normalized fpkm values? There are many discussions on this site over why not to use fpkm or rpkm values...

If you know what you are doing (and are sure that you need these values), you can use edgeR in R to do it.

ADD REPLYlink written 2.2 years ago by b.nota4.3k
1

Most often the conclusion is not to use fpkm and just use raw counts in a statistical framework like DESeq2 and edgeR.

ADD REPLYlink written 2.2 years ago by WouterDeCoster30k

we want to compare log2 male/female fpkm autosomal vs sex linked genes density distribution. for this we are comparing two different samples that differ in sample size (read counts).

is it preferred to go with raw counts or fpkm ?

ADD REPLYlink written 2.2 years ago by gangireddy160
1

If you are going to use edgeR or DESeq2 then you need raw counts, not normalized.

ADD REPLYlink written 2.2 years ago by WouterDeCoster30k
1
gravatar for EVR
2.2 years ago by
EVR470
Earth
EVR470 wrote:

hi,

Try using egdeR or Deseq2. take a look at trinity's pipeline for normalizatio which make use of edgeR's TMM normalization methid and outputs the TMM values.

ADD COMMENTlink written 2.2 years ago by EVR470

I have the same question as gangireddy, but how can you enter FPKMs in edgeR?, just as you would do with raw counts?, never mind the non-integers?

ADD REPLYlink written 2.1 years ago by lay_040

Use raw read counts!

ADD REPLYlink written 16 months ago by b.nota4.3k
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