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-   -   using Cuffdiff with biological replicates (http://seqanswers.com/forums/showthread.php?t=13805)

Jane M 09-01-2011 12:42 AM

using Cuffdiff with biological replicates
 
Hello,

I have seen several topics about Cuffdiff but I haven't found information about the use of Cuffdiff with biological replicates.

I try to use Cuffdiff to do analyze differentially expressed isoforms. I am studying 2 conditions (Raman - HM1) and there are 3 replicates per condition. There are 5295 genes and 6432 isoforms.
In the documentation, it's written "If you have more than one replicate for a sample, supply the SAM files for the sample as a single comma-separated list".
So I ran Cuffdiff this way:

Quote:

cuffdiff -L Raman,HM1 -N --FDR 0.05 /path/fichier.gtf /path/Cufflinks/L2/accepted_hits.bam,/path/Cufflinks/L4/accepted_hits.bam,/path/Cufflinks/L6/accepted_hits.bam /path/Cufflinks/L3/accepted_hits.bam,/path/Cufflinks/L7/accepted_hits.bam,/path/Cufflinks/L8/accepted_hits.bam
The replicates for the condition Raman are L2, L4 and L6. The replicates for the condition HM1 are L3, L7 and L8.
I'm interested in the output files isoform_exp.diff:

Code:

test_id        gene        locus        sample_1        sample_2        status        value_1        value_2        ln(fold_change)        test_stat        p_value        significant
EHI_000010.ref        -        DS571600:2419-3622        q1        q2        NOTEST        8.28385        21.4211        0.950069        -2.32208        0.0202284        no
EHI_000130.alt1        -        DS571600:7792-8309        q1        q2        OK        108.42        6.20207        -2.86113        4.25521        2.08856e-05        yes
EHI_000130.ref        -        DS571600:7792-8309        q1        q2        OK        1152.64        2299.79        0.690763        -16.1849        0        yes
EHI_000240.ref        -        DS571186:1554-2669        q1        q2        OK        558.654        857.323        0.428284        -7.87676        3.33067e-15        yes
EHI_000250.ref        -        DS571186:2850-3551        q1        q2        OK        134.444        301.066        0.80618        -7.77203        7.77156e-15        yes
...
EHI_C00159.ref        -        EH4264:45-392        q1        q2        NOTEST        11.8076        27.8643        0.858599        -2.4726        0.0134133        no
EHI_000010.ref        -        DS571600:2419-3622        q1        q3        NOTEST        8.28385        14.2453        0.542123        -1.24073        0.214705        no
EHI_000130.alt1        -        DS571600:7792-8309        q1        q3        OK        108.42        4.26615        -3.2353        4.30991        1.63319e-05        yes
EHI_000130.ref        -        DS571600:7792-8309        q1        q3        OK        1152.64        1736.74        0.409956        -9.25409        0        yes
...
EHI_C00155.ref        -        DS571588:780-994        q5        q6        OK        4382.71        4702.45        0.0704184        -3.35392        0.000796741        yes
EHI_C00156.ref        -        DS571646:5447-6016        q5        q6        NOTEST        12.2637        40.6463        1.19826        -3.67794        0.000235123        no
EHI_C00157.ref        -        DS571705:1482-1899        q5        q6        OK        776.201        1585.64        0.714329        -16.3066        0        yes
EHI_C00159.ref        -        EH4264:45-392        q5        q6        NOTEST        9.16532        4.74531        -0.65827        1.16396        0.244442        no

Here is my problem. The file contains 96480 (15*6432) lines (instead of 6432). 15 is the number of combinations between the 6 data sets... I think Cuffdiff did not consider the 3 biological replicates as one condition !
Moreover, I don't know why I don't have the adjusted p-value information...

How can I use Cuffdiff to get differentially expressed isoforms ?
Thanks for your help,
Jane


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