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  • Cuffdiff2 skipped genes

    Hi everyone,

    I have weird problem. I followed the Tuxedo pipeline it has been working well so far. I was conducting CummeRbund and in my heatmap visualization, I noticed one of the genes of interest was not present. I went back to all my Cufflinks and Cuffmerge folders and saw that the genes of interest or location of the gene was present there. But in cuffdiff2's gene_exp.diff file, it did not have gene loci present.

    For example it went from:

    Gene10355
    Gene10357

    Gene10356 was skipped.

    What did I do wrong to have one of my gene of interest missing in my gene_exp.diff.

    Here's my pipeline:

    Trimmomatic > Tophat 2 with Ensembl GTF and Fasta > Cufflinks 2.0.2 with Ensembl Fasta (fragment bias) and GTF > Cuffmerge2 2.0.2 with Ensembl GTF > Cuffdiff2 2.0.2 with Ensembl Fasta and merged_w_refs.gtf file from Cuffmerge2 2.0.2_Fragment(False discovery rate of 0.01 and multiple hits correction) > CummeRbund for heatmap

    Also I have 2 replicates for Organ A and 3 replicates for Organ B.

    Thank you in advance.

    EDIT: Looking at the Cufflinks files, the expression (FPKM) of the gene is high in Organ A and literally 0 in Organ B. Would this lead to this problem?

    EDIT2: I still not get the skipped gene even when I take out multi hit correction or taking away the fasta file.

    EDIT: In the Cuffmerge file: The first 2 matches are Cuff (Organ A) and the second 2 matches are the actual gene location (Organ B). Could that cause it not work properly for Cuffdiff2?

    This is very strange and I did not have this occur before. Could someone kindly help me on this. Thank you. I would really appreciate it.

    -Zapages
    Last edited by Zapages; 02-07-2014, 08:50 AM.

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