Question: differential expression with cuffdiff
0
gravatar for yaghoub.amraei
6 weeks ago by
yaghoub.amraei10 wrote:

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 • 175 views
ADD COMMENTlink modified 6 weeks ago by kristoffer.vittingseerup3.4k • written 6 weeks ago by yaghoub.amraei10
1
gravatar for JC
6 weeks ago by
JC11k
Mexico
JC11k wrote:

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

ADD COMMENTlink written 6 weeks ago by JC11k

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

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by yaghoub.amraei10

Yes, you can, but again, that is deprecated

ADD REPLYlink written 6 weeks ago by JC11k

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

ADD REPLYlink written 6 weeks ago by kristoffer.vittingseerup3.4k

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

ADD REPLYlink written 6 weeks ago by dsull1.5k

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

ADD REPLYlink written 4 weeks ago by kristoffer.vittingseerup3.4k
1

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.

ADD REPLYlink modified 4 weeks ago • written 4 weeks ago by dsull1.5k

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

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by ATpoint36k

my goal is LncRNA detection...

ADD REPLYlink written 5 weeks ago by yaghoub.amraei10

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

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

ADD REPLYlink written 4 weeks ago by dsull1.5k
1
gravatar for kristoffer.vittingseerup
6 weeks ago by
European Union
kristoffer.vittingseerup3.4k wrote:

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.

ADD COMMENTlink written 6 weeks ago by kristoffer.vittingseerup3.4k

Can you explain more about this or give an example?

ADD REPLYlink written 13 days ago by yaghoub.amraei10
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