[Bioc-sig-seq] using Cuffdiff with biological replicates

[Jane Merlevede]

Yes, sorry...
I was manipulating my rna-seq data with R and I forgot this mailing
list is for Bioconductor softwares only... Sorry

2011/9/1 Martin Morgan <mtmorgan at fhcrc.org>:
> On 09/01/2011 01:45 AM, Jane Merlevede wrote:
>>
>> Hello,
>>
>> I try to use Cuffdiff to do analyze differentially expressed isoforms.
>
> Hi Jane -- this mailing list is for Bioconductor software; please ask
> questions exclusively about cuffdiff elsewhere, e.g., SeqAnswers, Biostar,
> or at the link at http://cufflinks.cbcb.umd.edu/manual.html.
>
> Or are you looking for help using edgeR or DESeq ?
>
> Martin
>
>
>> 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:
>>
>> 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:
>>
>> 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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>
>
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