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  • cuffdiff 2.0.2 frag-bias-correct problem

    I encountered a strange problem while using cuffdiff 2 to analyze my rna-seq data.


    I used tophat 1.4.1 to map my rna-seq data to pig genome (susscorfa 10.2), then i ran DE analysis with cuffdiff (both v1.3 and v2.0.2).

    the command i used is as following (for both version of cuffdiff)

    cuffdiff --no-update-check -v -o diff_out_mask_upperN -M rRNA.gtf --library-type fr-secondstrand -N -u -b Sus_scrofa.Sscrofa10.2.68.dna.toplevel.fa -p 16 -L ConditionA,ConditionB Sus_scrofa.Sscrofa10.2.68.gtf ConditionA.bam ConditionB.bam


    Strangely, the result from cuffdiff v2.0.2 showed the expression of all transcripts is zero! (only show first couple of lines in gene_exp.diff)

    test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
    ENSSSCG00000000001 ENSSSCG00000000001 - chr5:588810-596477 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000002 ENSSSCG00000000002 GTSE1 chr5:544685-564101 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000003 ENSSSCG00000000003 TTC38 chr5:520893-543410 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000004 ENSSSCG00000000004 PKDREJ chr5:509043-516263 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000005 ENSSSCG00000000005 C22ORF40 chr5:496539-504125 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000006 ENSSSCG00000000006 PPARA chr5:424928-488440 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000007 ENSSSCG00000000007 TRMU chr5:569580-583238 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000010 ENSSSCG00000000010 FBLN1 chr5:1071719-1158793 Infa Remo NOTEST 0 0 0 0 1 1 no
    ENSSSCG00000000011 ENSSSCG00000000011 - chr5:1324339-1373617 Infa Remo NOTEST 0 0 0 0 1 1 no

    but the result from cuffdiff 1.3 showed most transcripts were expressed:

    ENSSSCG00000000001 ENSSSCG00000000001 - chr5:588810-596477 Infa Remo NOTEST 0.515866 0.815506 0.660699 -0.560886 0.574875 1 no
    ENSSSCG00000000002 ENSSSCG00000000002 GTSE1 chr5:544685-564101 Infa Remo NOTEST 2.95675 3.76521 0.348715 -0.75018 0.453146 1 no
    ENSSSCG00000000003 ENSSSCG00000000003 TTC38 chr5:520893-543410 Infa Remo NOTEST 2.32987 2.75627 0.242466 -0.429257 0.667736 1 no
    ENSSSCG00000000004 ENSSSCG00000000004 PKDREJ chr5:509043-516263 Infa Remo NOTEST 0.422011 0.45496 0.108459 -0.150356 0.880484 1 no
    ENSSSCG00000000005 ENSSSCG00000000005 C22ORF40 chr5:496539-504125 Infa Remo OK 3.54952 4.91868 0.470645 -0.744261 0.456718 0.882984 no
    ENSSSCG00000000006 ENSSSCG00000000006 PPARA chr5:424928-488440 Infa Remo OK 11.487 20.1291 0.809283 -2.13796 0.03252 0.415541 no
    ENSSSCG00000000007 ENSSSCG00000000007 TRMU chr5:569580-583238 Infa Remo NOTEST 2.27422 1.42395 -0.675472 1.22796 0.219461 1 no
    ENSSSCG00000000010 ENSSSCG00000000010 FBLN1 chr5:1071719-1158793 Infa Remo OK 41.1482 36.0558 -0.190598 0.593366 0.552936 0.906035 no
    ENSSSCG00000000011 ENSSSCG00000000011 - chr5:1324339-1373617 Infa Remo NOTEST 0 0 0 0 1 1 no

    when I removed frag-bias-correct parameter while analyzing DE with cuffdiff 2.0.2,

    cuffdiff --no-update-check -v -o merged_anno_bias_correct_no_scaffold_mask_upperN -M rRNA.gtf --library-type fr-secondstrand -N -u -p 16 -L ConditionA,ConditionB Sus_scrofa.Sscrofa10.2.68.gtf ConditionA.bam ConditionB.bam

    the result seemed much normal:

    ENSSSCG00000000001 ENSSSCG00000000001 - chr5:588810-596477 Infa Remo NOTEST 0.0163158 0.046151 1.50009 -1.16742 0.24304 1 no
    ENSSSCG00000000002 ENSSSCG00000000002 GTSE1 chr5:544685-564101 Infa Remo OK 0.0846721 0.181485 1.09989 -1.68917 0.0911874 0.474875 no
    ENSSSCG00000000003 ENSSSCG00000000003 TTC38 chr5:520893-543410 Infa Remo OK 0.0609228 0.119559 0.972666 -1.21118 0.225826 0.599345 no
    ENSSSCG00000000004 ENSSSCG00000000004 PKDREJ chr5:509043-516263 Infa Remo NOTEST 0.0112047 0.0208992 0.89934 -0.936853 0.348834 1 no
    ENSSSCG00000000005 ENSSSCG00000000005 C22ORF40 chr5:496539-504125 Infa Remo OK 0.131638 0.293968 1.15908 -1.36223 0.173124 0.554138 no
    ENSSSCG00000000006 ENSSSCG00000000006 PPARA chr5:424928-488440 Infa Remo OK 0.365693 1.11547 1.60895 -3.03111 0.00243654 0.298428 no
    ENSSSCG00000000007 ENSSSCG00000000007 TRMU chr5:569580-583238 Infa Remo NOTEST 0.0657846 0.0688074 0.0648129 -0.0842925 0.932824 1 no
    ENSSSCG00000000010 ENSSSCG00000000010 FBLN1 chr5:1071719-1158793 Infa Remo OK 1.23995 1.8773 0.598383 -1.33535 0.181762 0.562518 no
    ENSSSCG00000000011 ENSSSCG00000000011 - chr5:1324339-1373617 Infa Remo NOTEST 0 0 0 0 1 1 no


    what i don't get is that when using same genome sequence used in cuffdiff v1.3, why cuffdiff v2.0.2 reported the expression of all transcript are zero?

    Any comment would be appreciated. thanks.
    Last edited by johnwu; 03-12-2013, 11:41 PM.

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