getting RPKM and VST using DEseq2 package
2
0
Entering edit mode
7.0 years ago
Angel ★ 4.1k

Hello,

I read DEseq2 vignette but I could not find any function to RPKM and VST. Then how I can have these files?

Thank you

RNA-Seq R DEseq2 • 4.8k views
ADD COMMENT
1
Entering edit mode
7.0 years ago

Fereshteh

Don't stick to trying to calculate RPKM.

You can use other normalization methods included in DESeq2

In confused, you have choices

There are other packages, such as NOISeq, that allow you to normalize data through various methods. The nice thing about NOISeq is that it just after normalization, it will provide you with a statistical summary of each of the normalization method you try indicating if normalization is correct or not. You can then keep using NOISeq, or go to DESeq knowing what is the correct normalization

I did this with some of my data, and RPKM did not pass the filter. I had to try some other methods, and the latest I tried was OK

Give it a try. The NOISeq vignette will help you

ADD COMMENT
0
Entering edit mode
7.0 years ago

There's VST and RLog described on page 16 of the manual.

For RPKM, see Devon's answer here

Manual is available here

ADD COMMENT
0
Entering edit mode

thank you Andrew, But i read both already but no clue yet

if i open rstudio, which i should type to start the analysis?

ADD REPLY
1
Entering edit mode

You stated you've already read the vignette? - The DESeq2 manual is fantastically detailed and covers everything from preparing your counts / pheno table, and running all aspects of differential gene expression analyses. I'd suggest you go back and read the manual again, and follow it using example data provided in the package before starting on your own data.

ADD REPLY
0
Entering edit mode

thank you but nothing there about RPKM, also that was too puzzling

ADD REPLY
1
Entering edit mode

Why do you want RPKMs? If you look at the link I put in my answer, Devon explains how to calculate them along with a very apt warning about NOT using them with DESeq2.

ADD REPLY
0
Entering edit mode

thank you, but even i don't know how to normalize my raw counts

ADD REPLY
2
Entering edit mode

Ok, rule 1, if you don't know what to do, read the manual. The manual contains all the information about the tool, and even contains a guide with sample data to get your started if you're unsure. Frankly, I'm not going to spoon feed you the information from the manual.

ADD REPLY
0
Entering edit mode

yes you all right

thank you

ADD REPLY
0
Entering edit mode

Hey @andrew.j.skelton73, sorry to bump an old thread but I've got a related question. My understanding from the vignette and source code is rather that the the VST doesn't account for the feature length, but rather the inter-sample count variance by feature and the library size factors. Is this not the case? In using the normalized counts (from VST) for downstream analysis, is it not appropriate to adjust for feature lengths using the rpkm?

Thanks for your insight.

ADD REPLY
0
Entering edit mode

Check out this discussion on gene length in DESeq2.

ADD REPLY

Login before adding your answer.

Traffic: 1314 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6