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  • cuffdiff not performing splicing tests and promoter tests

    Hi all,

    I am using cufflinks package to look for differentially spliced exons. The pipeline I followed is tophat -> cufflinks -> cuffmerge/cuffcompare(tried with both) -> cuffdiff.
    Result on command line is like this :-

    Performed 23758 isoform-level transcription difference tests
    Performed 20313 tss-level transcription difference tests
    Performed 18306 gene-level transcription difference tests
    Performed 17903 CDS-level transcription difference tests
    Performed 0 splicing tests
    Performed 0 promoter preference tests
    Performing 0 relative CDS output tests
    Writing isoform-level FPKM tracking
    Writing TSS group-level FPKM tracking
    Writing gene-level FPKM tracking
    Writing CDS-level FPKM tracking
    Writing isoform-level count tracking
    Writing TSS group-level count tracking
    Writing gene-level count tracking
    Writing CDS-level count tracking
    Writing isoform-level read group tracking
    Writing TSS group-level read group tracking
    Writing gene-level read group tracking
    Writing CDS-level read group tracking
    Writing read group info
    Writing run info

    Command I used to run cuffdiff is :-
    ./cuffdiff --no-update-check -o day0 -p 2 -N -b /home/Vaibhav/Alt_transcriptome_data/ws230/ws230.fa -u merged_asm/merged.gtf /home/Vaibhav/Alt_transcriptome_data/new/again_X_day0/accepted_hits.bam /home/Vaibhav/Alt_transcriptome_data/new/again_Y_day0/accepted_hits.bam

    I have no replicates of the sample. Now I am getting some differential results for gene, cds and isoform but getting nothing for splicing and promoter something I am interested in. I also tried lowering c option till 2, but got the same result.Can someone please suggest me what should I do so that cuffdiff performs splicing tests.
    Really in need of the differential spliced exons for my data.

    Thanks a lot

  • #2
    hi vaibhav_jain,

    did you solve the problem? I having similar issues and can't for the life of me figure out what is going on.

    Comment


    • #3
      Hi, vaibhav_jain and krespim:
      Have you solved the problem? This has puzzled me almost one week. Why cuffdiff performed 0 splicing test?

      Comment


      • #4
        Originally posted by leifive View Post
        Hi, vaibhav_jain and krespim:
        Have you solved the problem? This has puzzled me almost one week. Why cuffdiff performed 0 splicing test?

        Not really a solution, but when I load the results in cuffdiff I do get DE isoforms. Do try that. Maybe it is just a bug in the reporting.

        Comment


        • #5
          Originally posted by krespim View Post
          Not really a solution, but when I load the results in cuffdiff I do get DE isoforms. Do try that. Maybe it is just a bug in the reporting.
          Thanks for your reply, krespim. But I don't think this is just a bug in the reporting, because there is no "OK" status in my splicing.diff file from Cuffdiff, but NOTEST or LOWDATA instead. And I have tried both combined.gtf and merged.gtf as input of cuffdiff, results are almost the same.

          Comment


          • #6
            Originally posted by leifive View Post
            Thanks for your reply, krespim. But I don't think this is just a bug in the reporting, because there is no "OK" status in my splicing.diff file from Cuffdiff, but NOTEST or LOWDATA instead. And I have tried both combined.gtf and merged.gtf as input of cuffdiff, results are almost the same.

            Well, I can't help much further but I can tell you what I end up doing. As I am working with species that have relative well annotated genomes/transcriptomes (Homo sapiens and Mus musculus), I forego the initial cufflinks step and go straight to cufdiff after mapping using the gtf provided in iGenomes as a reference. Here are my settings:

            # cuffdiff v2.0.2

            $cuffdiff -L "GFP,KD" -o $cuff_output -p 8 -u $gtfFile $gfp_a,$gfp_b $srsf1_a,$srsf1_b

            And that seems to work.

            Comment


            • #7
              Originally posted by krespim View Post
              Well, I can't help much further but I can tell you what I end up doing. As I am working with species that have relative well annotated genomes/transcriptomes (Homo sapiens and Mus musculus), I forego the initial cufflinks step and go straight to cufdiff after mapping using the gtf provided in iGenomes as a reference. Here are my settings:

              # cuffdiff v2.0.2

              $cuffdiff -L "GFP,KD" -o $cuff_output -p 8 -u $gtfFile $gfp_a,$gfp_b $srsf1_a,$srsf1_b

              And that seems to work.
              Thanks krespim. I have solved the problem by going back to Cufflinks 1.3.0, and I'm not sure whether there is a bug in the lastest version of Cufflinks or something incompatible in my reference file. However, it's done now.

              Comment


              • #8
                Update

                Originally posted by leifive View Post
                Thanks krespim. I have solved the problem by going back to Cufflinks 1.3.0, and I'm not sure whether there is a bug in the lastest version of Cufflinks or something incompatible in my reference file. However, it's done now.
                I went back to my notes - because I had to run cuffdiff again - and the problem seems to occur when the annotation gft is not a cufflinks output or from iGenomes (not 100% sure about iGenomes though). The reason is that to perform the splicing tests the gtf needs to have some extra annotated features.

                Solution: run cuffcompare in the orginal gtf.
                cuffcompare -s hg19.fasta -CG -r EnsemblGene-67.gtf EnsemblGene-67.gtf

                This will create a cuff* ready annotation file.

                Comment

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