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  • Cufflinks and Cuffdiff question on Galaxy

    I just looked through all of my Cufflink results, I only found my results to be in FPKM with high and low confidence values. Then I realized I did not conduct Cuffdiff, which required 2 bam files and .gtf or gff file. After importing the other replicate .bam file from NCBI and downloading the GTF file for the specie that I am working with from http://genome.ucsc.edu/cgi-bin/hgTables? . I then conducted Cuffdiff on Galaxy-project to get the table for the p and q value, but the values for p and q were all 1. This is very weird. I do not think every sequence value should 1.

    Am I doing something wrong?

    On galaxy-project for Cuffdiff, it requests the following:
    Transcripts: The Species1.GTF file
    BAM file of aligned RNA-Seq Reads: Species1_Replicate1.bam
    BAM file of aligned RNA-Seq Reads: Species1_Replicate2.bam

    or

    Transcripts: The Species1.GTF file
    BAM file of aligned RNA-Seq Reads: Species1_Replicate1_with_Organ.bam
    BAM file of aligned RNA-Seq Reads: Species1_Replicate1_without_Organ.bam

    Both of these result with the same result.

    Both of the BAM files have been aligned and indexed via Tophat and Bowtie on NCBI.

    Am I doing this correctly? Shouldn't the p value be something small like 0.01 or 0.001 as cuffdiff is like T test?

    Could anyone please help me. Thank you.

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