How to perform FPKM and TMM calculations?

Question

[k.kathirvel93]

Hi EveryOne,
I may think, this will be a silly question. But I am new to miRNA and RNA seq data analysis. I am working in human data, Mapping and quantification has done already, now am in normalization process. I knew some backgrounds about what is FPKM and RPKM, TMM and Quantile. But i want to get to know about how to perform these normalization methods. Which language or which tool, and How ? Thanks in advance.

[WouterDeCoster]

If your aim is to perform differential expression analysis then you should not do any normalization yourself. You should use the raw read counts in a tool such as DESeq2.

[k.kathirvel93]

Thanks WouterDeCoster, Yes am looking for differential expression. But many RNA-seq papers coming with Normalization. I am so confused.

[WouterDeCoster]

FPKM is a very simple normalization approach. DESeq2 (and others such as edgeR) use a much more sophisticated method to normalize your read counts. So normalization has to be done, but the software will do it for you and you don't need to worry about it.

[k.kathirvel93]

Thanks WouterDeCoster, Can you clarify my doubts: 1.DESeq2 will do ? and 2. edgeR will do TMM?

[WouterDeCoster]

The documentation and publications of those tools should tell you everything you want to know about them.

[k.kathirvel93]

Yes WouterDeCoster, Can I get the instructions and protocols of every of them. If you can.

[k.kathirvel93]

Hi WouterDeCoster, Can you suggest me which normalization method is best for miRNA-seq data and RNA-seq data ? Thanks in advance.

[Sej Modha]

You can read the post I have tagged below to get more info about the best method for miRNA-seq normalisation.

Answer 1

[Sej Modha]

A number of tools such as edgeR and Deseq have normalisation methods built-in. More info: Best Way To Measure Mirna-Seq Abundance