Cuffdiff with 3 conditions

Question

[pablo]

Hi guys,

I want to run Cuffdiff on my Tophat alignment files.

I have 6 samples : 3 conditions (A,B,C) with 2 replicates each . I'd like to compare each condition between each other (A vs B, A vs C, B vs C)

I tried

cuffdiff -L c1,c2,hs1,hs2,rv1,rv2 \
    -p 32 ../Araport11_GFF3_genes_transposons.201606_filtred.gff \
    -o cuffdiff/ \
    SRR2033948/accepted_hits.bam,SRR2033949/accepted_hits.bam,SRR2033950/accepted_hits.bam,\
    SRR2033951/accepted_hits.bam,SRR2033952/accepted_hits.bam,SRR2033953/accepted_hits.bam

But that doesn't return me what I want

Any suggestion?

Best, Vincent

[Ram]

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.

Answer 1

[i.sudbery]

CuffDiff is outdated software and is no longer best practice (if it ever was). I personally recommend salmon or kallisto followed by tximport and DESeq2, although salmon/kallisto followed by tximport and limma-voom or edgeR or salmon/kallisto followed by sleuth also all work well. If you are only interested in genes, you can also get away with mapping followed by featureCounts then any of DESeq2, limma-voom or edgeR.

If you must use CuffDiff, your input should look like:

cuffdiff -L c,hs,rv \
    -p 32 ../Araport11_GFF3_genes_transposons.201606_filtred.gff \
    -o cuffdiff/ \
    SRR2033948/accepted_hits.bam,SRR2033949/accepted_hits.bam \
    SRR2033950/accepted_hits.bam,SRR2033951/accepted_hits.bam \
    SRR2033952/accepted_hits.bam,SRR2033953/accepted_hits.bam