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Old 10-17-2011, 01:37 AM   #2
zeam
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Location: USA

Join Date: Oct 2010
Posts: 38
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For question 1, did you use an annotation in cufflinks or cuffcompare? If so ,I thinks this might explain this:
Cuffdiff and Cufflinks now accept new options controlling whether all hits are counted towards the FPKM denominator, or only those compatible with some transcript in the reference annotation. Counting only compatible hits avoids certain types of bias that arise when one sample contains far more hits that aren't compatible with any transcript than the other sample does. For example, if one sample contains vastly more mapped ribosomal RNA hits, FPKM values will appear lower in that sample, potentially leading to false positive differential expression calls. Cuffdiff by default now uses only compatible hits. Cufflinks still uses total hits by default, as using compatible hit accounting requires a reference GTF.
So just set --compatible-hits-norm parameter to be identical when you do cufflinks and cuffdiff.

Also,I have one question, you said you have three bam files, A B and C.Did you use cuffdiff for each pair or just use cuffdiff once? If the later, did you get all cuffdiff test between all pairs of samples ? I have four tisssues, and command are as follows:
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cuffdiff -o DEG_cuffdiff -p 20 cuffcmp.combined.gtf ./tophat_sd/accepted_hits.bam ./tophat_em/accepted_hits.bam tophat_en1/accepted_hits.bam ./tophat_en2/accepted_hits.bam
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But I only got one cuffdiff test file between the fisrt two pairs? Follow the mannual, there should be 6 files for each pair of samples.
Thanks.
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