RUnning Cuffdiff on 2 conditions simultaneously
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5.4 years ago

I have two sets of data; 12 hours control and experiment and 48 hours control and experiment. I am only interested in known transcripts so I will be using cuffdiff directly. My question is how do I properly combine the controls and each time period as replicate groups in cuffdiff? Do I create a group for each time period and just add the control and experiment in batch mode, or do I add the control and experiment as individual replicates in each group?

In addition, If I have a sample with two replicates and a control with two replicates do I add both controls and both experimental replicates to individual groups?

RNA-Seq differential expression Assembly • 1.3k views
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Entering edit mode
5.4 years ago
Benn 8.1k

For your first question, according to the manual (http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/), cuffdiff compares all samples pairwise. So choose afterwards what relevant contrast you want to have. There is btw also a time series option.

I do not exactly understand your second question, can you be more specific about what you mean by replicate? Do you mean technical replicate?

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The second question I figured out. With the time series I am still a little confused. So I would put in the command line 12 hours.bam \ control1.bam \ 48hours.bam \ control2.bam and it would compare each to the control. I am not looking to compare expression between two time points, but would like to have some indication that the log2 values it produces are correct since for this sample I do not have any technical replicates.

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