differential expression with cuffdiff
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17 months ago

Hi all. I have 24 samples RNA-seq, paired-end data including 2 genotypes, 3 Zone from root, 2 conditions and each condition including two replicate. How can I do cuffdiff for this expriments. thanks alot

RNA-Seq • 535 views
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17 months ago
JC 12k

Cuffdiff is no longer recommended, update your analysis to HISAT / StringTie / Ballgown

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Thanks, Can't use Caddiff with this number of samples?

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Yes, you can, but again, that is deprecated

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Were did you hear/read that Cufflinks/Cuffdiff is deprecated?

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The authors of those tools say so themselves (see their lab websites; their twitters; their previous posts on biostars; etc.).

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I have not been able to find it. Could you point to something more recent than this (which states cufflinks it is not deprecated).

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Sure.

Most recent: This mentions it as "retired software" and "no longer under active development and are not being supported". It does not list it as "now maintained/developed elsewhere".

Older posts: See this tweet (referring to it as obsolete). And see this.

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I would also not use this pipeline. Stringtie is overly complicated unless you want to assemble the transcriptome. For a standard DE analysis (i a proper reference transcriptome exists) there is (imho) no need to bother with it. ALso leightweight pseudo- or selective aligners such as kallisto and salmon typically outperform traditional alignment for RNA-seq quantification (check any of the recent benchmark papers). This ballgown tool does not really provide features which other, better documented and actively maintained tools such as DESeq2 or edgeR do not offer.

I would go with quantification by salmon, importing data into R with tximport, then DESeq2, as described e.g. here: https://www.bioconductor.org/packages/devel/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html

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my goal is LncRNA detection...

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Recommendations for lncRNA: https://academic.oup.com/gigascience/article/8/12/giz145/5663671

Still recommend pseudoalignment, e.g. kallisto, for this.

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17 months ago

Unfortunately you cannot analyse your experimental setup with Cuffdiff as it cannot handle overlapping conditions/co-factors. For this you need to use cuffquant and cuffnorm (see overview here) to get a count matrix which you can then analyze with DESeq2, edgeR or similar.

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Can you explain more about this or give an example?

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