Hi Thanh,

This is a question about Cuffdiff, rather than about Galaxy directly.  Try
posting the question to one of these forums:

  https://groups.google.com/forum/#!forum/tuxedo-tools-users
  http://seqanswers.com/forums/forumdisplay.php?f=18 - Bioinformatics forum
  http://seqanswers.com/forums/forumdisplay.php?f=26 - RNA sequencing forum
  http://www.biostars.org/

Thanks,

Dave C




On Fri, Jul 19, 2013 at 4:56 PM, Hoang, Thanh <hoan...@miamioh.edu> wrote:

> Hi all,
> I have some questions about how Cufffdiff does the  statistical analysis.
> I am looking for DE genes in two sample groups ( 3 replicate per group).
> In the Cuffdiff;s gene_exp.diff, I found many genes that have very large
> RPKM fold-change between two groups (with p value < or > 0.05)  but still
> NO significant. Something like this:
>
> test_id gene_id gene locus sample_1 sample_2 status value_1 value_2
> log2(fold_change) test_stat p_value q_value significant
> ENSMUSG00000047139 ENSMUSG00000047139 Cd24a 10:43579168-43584262 q1 q2 OK
> 96.2585 2700.55 4.8102 1.6486 0.03995 0.078237 no  ENSMUSG00000066975
> ENSMUSG00000066975 Cryba4 5:112246492-112252518 q1 q2 OK 424.582 46190.2
> 6.7654 0.598327 0.3408 0.442128 no
>
> Then I checked the READ_GROUP_TRACKING file for those genes to check the
> RPKM value for each replicate:
>
>      tracking_id condition replicate raw_frags internal_scaled_frags
> external_scaled_frags FPKM effective_length status  ENSMUSG00000047139 q1
> 1 11256 5876.82 5876.82 125.915 - OK  ENSMUSG00000047139 q1 0 3783 4343.44
> 4343.44 42.0316 - OK  ENSMUSG00000047139 q1 2 10051 5639.48 5639.48
> 120.829 - OK  ENSMUSG00000047139 q2 1 76771 156059 156059 3343.66 - OK
> ENSMUSG00000047139 q2 0 82394 162172 162172 1420.33 - OK
> ENSMUSG00000066975 q1 1 12825 6696 6696 407.899 - OK  ENSMUSG00000066975
> q1 0 3694 4241.26 4241.26 375.211 - OK  ENSMUSG00000066975 q1 2 14397
> 8077.95 8077.95 490.636 - OK  ENSMUSG00000066975 q2 1 348103 707619 707619
> 42455.1 - OK  ENSMUSG00000066975 q2 0 420896 828430 828430 48920.6 - OK
> ENSMUSG00000066975 q2 2 331098 767405 767405 47195 - OK
>
>
> Would not I expect these DE genes are significant? Could anyone explain
> why  Cufflinks show  this result?
>
>
> Best regards
> Thanh
>
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