Question: Is Cufflinks2's normalization method reliable for differential expression analysis?
gravatar for kevin.l.yang
3.1 years ago by
kevin.l.yang0 wrote:

I am using both DESeq2 and Cufflinks2 for differential expression analysis for some mouse RNAseq data. I was reading this guide ( for help. Page 49 of the guide says that RPKM/FPKM isn't suitable for differential expression analysis, since it doesn't take into account sequencing depth among different samples. However, I figured that going from Cufflinks to Cufflinks2, the developers would have accounted for this issue. Can I trust my results from Cufflink2? I am making a poster right now, and I don't want to put my Cufflinks2 results if they're not reliable

rna-seq • 859 views
ADD COMMENTlink modified 3.1 years ago by Kevin Blighe66k • written 3.1 years ago by kevin.l.yang0
gravatar for Kevin Blighe
3.1 years ago by
Kevin Blighe66k
Kevin Blighe66k wrote:

You're right: for differential expression itself, I don't believe that Cufflinks is ideal due to the fact that it doesn't handle the wide variation in counts that can exist. One can end up with log base 2 fold change difference of upward of 100, which is astronomical and doesn't make much sense. You are not required to use FPKM with Cufflinks, though; If you must use Cufflinks, for whatever reason, then use geometric normalisation.

DESeq2 handles these issues very well and allows you to perform a regularised log transformation (rlog) or variance stabilised transformation (VST) on your data, which produces very nice P values and fold-changes. So, many people then try to extract raw counts from the TopHat/Cufflinks method and then do the differential expression in DESeq2, but this is not possible directly using TopHat/Cufflinks.

A much simpler way to analyse RNA-seq data is with Kallisto ( ), which extracts raw counts over a CDS FASTA that you supply. If you supply GENCODE's CDS FASTA ( ), then you can get count abundances over upward of 200,000 transcripts and their isoforms.

ADD COMMENTlink modified 21 months ago • written 3.1 years ago by Kevin Blighe66k
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