Question: Cuffdiff promoter.diff is empty
0
gravatar for Satyajeet Khare
4.3 years ago by
Satyajeet Khare1.6k
Pune, India
Satyajeet Khare1.6k wrote:

Hi Biostars,

I performed Cuffdiff between two samples with three replicates each. While gene_exp.diff, cds_exp.diff and splicing.diff files are filled with entries, promoter.diff and isoform_exp.diff files are empty. This is a bit unusual. Here is the command for cuffdiff

cuffdiff --use-sample-sheet -C contrasts.txt -p 10 -o ../cuffdiff ../cuffmerge/merged.gtf sample_sheet.txt

merged.gtf was produced using following command

cuffmerge -o ../cuffmerge/ -g annotation.gtf -s assembly.genome.fa -p 10 assembly_GTF_list.txt

This happened to two consecutive datasets. I am thinking of reinstalling Cufflinks. Any other suggestions?

Best,

rna-seq cuffdiff • 1.3k views
ADD COMMENTlink modified 4.3 years ago • written 4.3 years ago by Satyajeet Khare1.6k

No luck even after reinstallation

Performed 0 isoform-level transcription difference tests
Performed 0 tss-level transcription difference tests
Performed 14140 gene-level transcription difference tests
Performed 13567 CDS-level transcription difference tests
Performed 5386 splicing tests
Performed 0 promoter preference tests
Performing 0 relative CDS output tests

Here is a kind of result I am talking about. No promoter preference test or tss-level transcription difference test performed.

Still looking for solutions.

ADD REPLYlink written 4.3 years ago by Satyajeet Khare1.6k

can you provide the content of contrasts.txt?

ADD REPLYlink written 3.8 years ago by Malcolm.Cook1.1k
1
gravatar for Satyajeet Khare
4.3 years ago by
Satyajeet Khare1.6k
Pune, India
Satyajeet Khare1.6k wrote:

Found the answer. Quite unexpected one.

If I do not use sample sheet and write an elaborate command as follows, it works!

cuffdiff -p 10 -o cuffdiff/ merged.gtf -L Wt,KO ../tophat2/wt1/accepted_hits.bam,../tophat2/wt2/accepted_hits.bam,../tophat2/wt3/accepted_hits.bam ../tophat2/ko1/accepted_hits.bam,../tophat2/ko2/accepted_hits.bam,../tophat2/ko3/accepted_hits.bam

Here is the output

Performed 29720 isoform-level transcription difference tests
Performed 21535 tss-level transcription difference tests
Performed 14140 gene-level transcription difference tests
Performed 13567 CDS-level transcription difference tests
Performed 5386 splicing tests
Performed 4847 promoter preference tests

If its not a mistake in my sample sheet or contrasts file, it could be a bug. It seems contrast file for single comparison does not work that well. Hope it helps others :)

ADD COMMENTlink modified 4.3 years ago • written 4.3 years ago by Satyajeet Khare1.6k
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