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 <hoangtv@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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