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  • edge
    Senior Member
    • Sep 2009
    • 199

    Inquiry about using Cuffdiff for multisample analysis

    Hi,

    I have 10 RNA-seq samples now.
    Samples Times (hours after inoculation)
    Susceptible NIL 0
    Susceptible NIL 3
    Susceptible NIL 6
    Susceptible NIL 9
    Susceptible NIL 12
    Resistant NIL 0
    Resistant NIL 3
    Resistant NIL 6
    Resistant NIL 9
    Resistant NIL 12

    My main objective is to identify and characterize resistant genes associated to disease/bacteria-infection in my plant samples.
    I have a reference genome and reference annotation of my plant samples.
    From my RNA-seq data, I don't have any biological replicate or technical replicate.

    As shown in cufflink link, http://cufflinks.cbcb.umd.edu/tutorial.html
    I have run the cuffdiff as shown in the "Differential analysis with gene and transcript discovery" section.
    cuffdiff merged_asm/merged.gtf S_NIL_0.bam S_NIL_3.bam S_NIL_6.bam S_NIL_9.bam R_NIL_0.bam R_NIL_3.bam R_NIL_6.bam R_NIL_9.bam

    From the above command, it generates a list of output file :
    var_model.info
    tss_groups.read_group_tracking
    tss_groups.fpkm_tracking
    tss_groups.count_tracking
    tss_group_exp.diff
    splicing.diff
    run.info
    read_groups.info
    promoters.diff
    isoforms.read_group_tracking
    isoforms.fpkm_tracking
    isoforms.count_tracking
    isoform_exp.diff
    genes.read_group_tracking
    genes.fpkm_tracking
    genes.count_tracking
    gene_exp.diff
    cds.read_group_tracking
    cds.fpkm_tracking
    cds_exp.diff
    cds.diff
    cds.count_tracking
    bias_params.info

    Do anybody has similar experience to use Cuffdiff for multisample analysis ?
    I hope can plot some PCA plot, heat-map, MA-plot, etc in order to identify and characterize resistant genes associated to disease/bacteria-infection in my plant samples.

    Below is one of the journal I refer so far, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223079/
    I have a similar experiment design as the journal above.
    But I have no much idea how to use Cuffdiff output for further analysis.

    Thanks for any advice.

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