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  • cuffcompare or cuffmerge for cuffdiff

    Hi ,all
    This is an old topic in our community.see here and here
    although C.Tapnell recommend cufflinks->cuffmerge->cuffdiff flow for diff exp analysis in hereand this new paper ,I must bring it again,beacause Too much confusion.

    I have 3 pair-end samples and hava two targets:
    [1] discovery new isoform and there structure
    [2] differential gene and transcript exp anlalysis and there structure
    tophat+cufflinks has no problem for 3 samples.

    for the this two aim.I use coffcompare analyze the transfrags which cufflinks assempled and cuffdiff analyze diff exp.

    one flow:
    cuffcompare -o compare -s genomic_seq.fa -r known.gtf tanscriptA.gtf transcriptB.gtf transcriptC.gtf
    cuffmerge -g known.gtf -s genomic_seq.fa 3_assembly_GTF_list.txt
    cuffdiff -o -b genomic_seq.fa -L A,B,C -u -p 6 merged.gtf A.bam B.bam C.bam
    I use cuffcompare,because cuffcompare output .refmap and tmap for each sample. I can extract every cuff_transcript's ref_gen and region from cufflinks result transcripts.gtf like this:
    for transcript ENSMUST00000048860
    IN sample A:
    Gene_name Transcript_id Class_code Cufflinks_transcript_id FPKM Coverage Transcript_length Ref_Transcript_length Chromosome Strand Start End Exon_num Exon_start-Exon_end;ditto
    Mreg ENSMUST00000048860 c Sample_A.442.1 9.998678 41.470300 243 2493 1 . 72205812 72206054 1 72205812-72206054;
    Mreg ENSMUST00000048860 = Sample_A.444.1 25.753304 108.941130 1695 2493 1 - 72206430 72258706 5 72206430-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72258591-72258706;
    In sample B:
    Mreg ENSMUST00000048860 j Sample_B.478.1 0.355742 1.460597 1682 2493 1 - 72206370 72243058 5 72206370-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72243016-72243058;
    Mreg ENSMUST00000048860 = Sample_B.478.2 1.652110 6.783196 1742 2493 1 - 72206370 72258693 5 72206370-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72258591-72258693;
    and in compare.tracking
    TCONS_00001024 XLOC_000542 Mreg|ENSMUST00000048860 c q1:Sample_A.442|Sample_A.442.1|100|9.998678|4.225939|15.771418|41.470300|- - -
    TCONS_00001025 XLOC_000542 Mreg|ENSMUST00000048860 = q1:Sample_A.444|Sample_A.444.1|100|25.753304|24.064335|27.442272|108.941130|1695 q2:Sample_B.478|Sample_B.478.2|100|1.652110|1.194891|2.109330|6.783196|1742 -
    TCONS_00002413 XLOC_000542 Mreg|ENSMUST00000048860 j - q2:Sample_B.478|Sample_B.478.1|22|0.355742|0.086913|0.624571|1.460597|- -
    and this gene in cuffdiff result(treated):
    Tracking_id Gene_id Gene_name Class_code Nearest_ref_id TSS Locus Sample_1 Sample_2 FPKM_1 FPKM_2 Foldchange log2(fold_change) test_stat p_value q_value Significant
    TCONS_00004275 XLOC_001277 Mreg j ENSMUST00000048860 TSS2418 1:72205806-72258881 sample_A sample_B 8.99002 0.315128 0.0350531 -4.83432 3.98775 6.67042e-05 0.00727599 yes
    see if I foucus ENSMUST00000048860 due to cuffdiff result based foldchange.I need back compare result find this known transcript matched cufflinks assembled transcripts result to decide the assembled transcripts is known(class code = or c) or novel(class code j).
    But the cuffdiff id TCONS_00004275 is not same with cuffcompare TCONS_id and the Locus 1:72205806-72258881 also not same. This make me couldnot find interest ENSMUST00000048860's nearest structure in sample A and SampleB.IS Sample_A.442.1 or Sample_A.444.1 or other?


    so I change the workflow (without cuffmerge):

    cuffcompare -o compare -s genomic_seq.fa -r known.gtf tanscriptA.gtf transcriptB.gtf transcriptC.gtf
    cuffdiff -o -b genomic_seq.fa -L A,B,C -u -p 6 combined.gtfA.bam B.bam C.bam
    also use example ENSMUST00000048860
    for compare result(treated):
    IN sample A:
    Mreg ENSMUST00000048860 c Sample_A.443.1 9.998678 41.470300 243 2493 1 . 72205812 72206054 172205812-72206054;
    Mreg ENSMUST00000048860 = Sample_A.444.1 25.753304 108.941130 1695 2493 1 - 72206430 72258706 572206430-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72258591-72258706;
    IN sample B:
    Mreg ENSMUST00000048860 j Sample_B.479.1 0.355742 1.460597 1682 2493 1 - 72206370 72243058 572206370-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72243016-72243058;
    Mreg ENSMUST00000048860 = Sample_B.479.2 1.652110 6.783196 1742 2493 1 - 72206370 72258693 572206370-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72258591-72258693;
    another confused,same cufflink+cuffcompare program but the cuff_id is diff ,Sample_A.442.1 Sample_A.444.1 with Sample_A.443.1 Sample_A.443.1 also in Sample_B

    in compare.tracking
    TCONS_00001024 XLOC_000542 Mreg|ENSMUST00000048860 c q1:Sample_A.443|Sample_A.443.1|100|9.998678|4.225939|15.771418|41.470300|- - -
    TCONS_00001025 XLOC_000542 Mreg|ENSMUST00000048860 = q1:Sample_A.444|Sample_A.444.1|100|25.753304|24.064335|27.442272|108.941130|1695 q2:Sample_B.479|Sample_B.479.2|100|1.652110|1.194891|2.109330|6.783196|1742 -
    TCONS_00002413 XLOC_000542 Mreg|ENSMUST00000048860 j - q2:Sample_B.479|Sample_B.479.1|22|0.355742|0.086913|0.624571|1.460597|- -
    TCONS_00003426 XLOC_000542 Mreg|ENSMUST00000048860 c - - q3:Sample_C.463|Sample_C.463.1|100|2.478294|1.853823|3.102766|10.598946|-
    TCONS_00003427 XLOC_000542 Mreg|ENSMUST00000048860 c - - q3:Sample_C.464|Sample_C.464.1|100|2.878927|1.557985|4.199870|11.712125|-
    this gene in cuffdiff result(treated):
    TCONS_00001025 XLOC_000542 Mreg = ENSMUST00000048860 TSS1655 1:72206327-72258693 sample_A sample_B 18.4693 1.30708 0.0707704 -3.82072 3.82424 0.000131174 0.0148275 yes

    the ENSMUST00000048860 TCONS_00001025 is same as one of comcompare TCONS_id and i konw it mapped Sample_A.444.1 and Sample_B.479.2. then i can find Sample_A.444.1 and Sample_B.479.2 structure
    Strand Start End Exon_num Exon_start-Exon_end;ditto
    - 72206430 72258706 5 72206430-72207593;72208896-72209059;72210646-72210736;72238617-- - 72238776;72258591-72258706;
    - 72206370 72258693 5 72206370-72207593;72208896-72209059;72210646-72210736;72238617-72238776;72258591-72258693;
    Then i can do next analysis

    but from this two flow the cuffdiff result are very different about this trascript ENSMUST00000048860
    cuffdiff result(treated):
    Tracking_id Gene_id Gene_name Class_code Nearest_ref_id TSS Locus Sample_1 Sample_2 FPKM_1 FPKM_2 Foldchange log2(fold_change) test_stat p_value q_value Significant
    TCONS_00004275 XLOC_001277 Mreg j ENSMUST00000048860 TSS2418 1:72205806-72258881 sample_A sample_B 8.99002 0.315128 0.0350531 -4.83432 3.98775 6.67042e-05 0.00727599 yes
    TCONS_00001025 XLOC_000542 Mreg = ENSMUST00000048860 TSS1655 1:72206327-72258693 sample_A sample_B 18.4693 1.30708 0.0707704 -3.82072 3.82424 0.000131174 0.0148275 yes
    whatever class code,fpkm,foldchange,and also there are other diff between two pipeline. no same known transcrips in the two cuffdiff result.
    I want to know which cuffdiff result is more credible,and how workflow can meet the needs of my analysis.

    Thanks
    Shen
    Last edited by upper; 05-08-2012, 12:39 AM.

  • #2
    Hi Shen,

    when you use cuffmerge, do you see any skipped regions with mouse?
    Have you checked the transcript lengths of the new assembly?
    I have seen in my output extremely long merged genes which were in fact severel different refseq IDs.

    Comment


    • #3
      Originally posted by Kcornelius View Post
      Hi Shen,

      when you use cuffmerge, do you see any skipped regions with mouse?
      Have you checked the transcript lengths of the new assembly?
      I have seen in my output extremely long merged genes which were in fact severel different refseq IDs.
      Hi Kcornelius,

      you mean the merged.gtf that cuffmerge output?
      I check some transcript, but almost in know region. can you show a example.

      Comment


      • #4
        Sure,

        I have posted one in a related thread:

        Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc

        Comment


        • #5
          Originally posted by Kcornelius View Post
          Sure,

          I have posted one in a related thread:

          http://seqanswers.com/forums/showthread.php?t=19533
          Hi Kcornelius,

          Yes, I found so long transcripts and it seems multiple loci are merged into a single locus when using cuffmerge!!

          I am totally confused whether to use cuffmerge or cuffcompare to merge assemblies from different experiemntal conditions.

          Comment


          • #6
            I think one alternative method is merging all the bam together, and run cufflinks once.
            Or, do in silico normalization for the large fastq files befor running tophat.

            Comment


            • #7
              Originally posted by ravipatel4 View Post
              Hi Kcornelius,

              Yes, I found so long transcripts and it seems multiple loci are merged into a single locus when using cuffmerge!!

              I am totally confused whether to use cuffmerge or cuffcompare to merge assemblies from different experiemntal conditions.
              Did you get the long merged loci even after specifying a reference gff to cuffmerge and using also reference based assemblies for cufflinks (providing -g gff to cufflinks)?

              Comment

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