Cuffdiff promoter.diff is empty

Question

[Satyajeet Khare]

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,

[Satyajeet Khare]

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.

[Malcolm.Cook]

can you provide the content of contrasts.txt?

Answer 1

[Satyajeet Khare]

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 :)