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  • multiple lines per single gene in gene_exp.diff output of cuffdiff

    question that was posted on another forum, maybe someone here will have an answer:

    after running cuffdiff, in the gene_exp.diff file there are multiple lines for the same gene, with different FPKM values.
    any idea for the cause or how should I handle this to receive a single value per gene?

    http://seqanswers.com/forums/showthr...4650#post34650

    thanks
    Reut

  • #2
    Reut,

    Have you gone back to your something.combined.gtf file to look up the entry by XLOC value?

    I assumed that this was a bug and wrote a little program that compares the gene_exp.diff file and the combined.gtf and fixes the locus field in gene_exp.diff to what it should be based on the gtf file.

    Has anyone else had any experience with this?

    Sam

    Comment


    • #3
      Multiple entries of a gene/locus at Cuffdiff Differential Gene Expression Output

      Hi,

      I'm also getting this probelm (gene_exp.diff):

      XLOC_021564 Hnf4a 3:165976692-166162683
      XLOC_022796 Hnf4a 3:165976692-166162683
      XLOC_022797 Hnf4a 3:165976692-166162683

      When I looked at isoform_exp.diff file for Hnf4a all the mentioned isoforms like in the same region 3:165976692-166162683, but seem to be distributed among the above locus ids such that no locus id above share any isoform:
      test_id gene_id gene locus
      TCONS_00057332 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00057337 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00057338 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00057339 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00057340 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00057341 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00057342 XLOC_021564 Hnf4a 3:165976692-166162683
      TCONS_00060713 XLOC_022796 Hnf4a 3:165976692-166162683
      TCONS_00060714 XLOC_022797 Hnf4a 3:165976692-166162683
      TCONS_00060715 XLOC_022797 Hnf4a 3:165976692-166162683

      My pipeline:

      Gsnap (Reference guided Mapping, default settings) --> Remove softclip bases (script shared by rna-star author Alexander Dobin) --> Cufflinks (Reference guided; default settings) --> Cuffmerge (all samples cufflinks transcript.gtf + reference.gtf) --> Cuffdiff (default settings)

      The reason to remove soft-clip bases prior to running cufflinks is to have compatibility of Gsnap mapping output with Cufflinks because cufflinks uses soft-clipped bases to do assembly leading to assembled transcripts sometimes beyond reference length and throws an error at Cuffmerge. For more info on this


      Right now I am re-running the above pipeline with TopHat but I don't think it will solve this problem, because the problem is at cuffmerge, I think, in deciding to include some transcript/isoform assembly under one locus id and some in the other, within the same locus region, leading to redundancy in locus region and gene enteries.

      This is not for 1 gene above. Overall in gene_exp.diff file:

      Out of 25667 genes 3776 are repeated
      Out of 23862 loci positions (format: chr:start-end) 5759 are repeated.


      That's a significant number of genes have multiple entries.

      Did anyone else have this problem as well?

      Cufflinks version I used: cufflinks/2.2.1
      Genome: Rat genome rn5 (Ensembl db)
      Reference gtf of genes: Ensembl database version 77 for rn5


      Thanks,
      Rahil
      Last edited by rahilsethi; 03-09-2015, 04:29 AM.

      Comment

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