Question: Cufflinks bias correction and DESEq2
0
gravatar for as9309
3.2 years ago by
as930920
as930920 wrote:

I ran all my RNA Seq data through galaxy to get FPKM files from original fastq data, but I did bias correction in Cufflinks. However, the DESeq2 manual stresses that you should use raw integer counts, not normalised counts. Does cufflinks normalise the data to some extent during bias correction? Should I have not performed bias correction before doing the DESEq2 analysis?

Thanks.

bias deseq2 cufflinks • 2.1k views
ADD COMMENTlink modified 3.2 years ago by Nicolas Rosewick7.5k • written 3.2 years ago by as930920

as you wrote, DESeq2 works with raw integer counts. this is something different from FPKM values (corrected or not). try using featureCounts or HTSeq-count for it.

ADD REPLYlink written 3.2 years ago by Martombo2.4k

Hi, thanks - do you know if there is software on R to do this? I believe HTSeq-count requires python and featureCounts (rsubread) is only compatible with Mac?

ADD REPLYlink written 3.2 years ago by as930920

Do you mean Windows R? I am afraid not, Rsubread only works in R on mac or linux.

You might wanna install a VM for linux?

ADD REPLYlink written 3.2 years ago by Benn6.6k

you might try easyRNAseq

ADD REPLYlink written 3.2 years ago by Martombo2.4k

This question about how to use cufflink data in DEseq2, edgeR, or limma has been asked so many times before. Do posters even look for answers before asking here?

ADD REPLYlink written 3.2 years ago by Benn6.6k
1
gravatar for andrew.j.skelton73
3.2 years ago by
London
andrew.j.skelton735.6k wrote:

Use the following route to do a DESeq2 Analysis: Align fastq files to your reference genome using your favourite splice aware aligner (STAR is probably best), Count using HTSeq_Count, or RSubread, then read that data into DESeq2. 

Do not use any output from Cufflinks or the Tuxedo pipeline as a whole to feed into DESeq2, that breaks major assumptions of the data. Tuxedo can produce Pseudo Counts, and that's probably the closest thing you'll get, but even then, don't use them to feed into DESeq2.

ADD COMMENTlink written 3.2 years ago by andrew.j.skelton735.6k
0
gravatar for Carlo Yague
3.2 years ago by
Carlo Yague4.4k
Belgium
Carlo Yague4.4k wrote:

Yes, Cufflink does bias correction on FPKM... but FPKM are not raw integer counts in the first place !

To get the raw counts, you'll need to use either FeatureCounts or HTseq-counts, not Cufflink.

ADD COMMENTlink modified 3.2 years ago • written 3.2 years ago by Carlo Yague4.4k
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