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I have just loaded the latest CummeRbund program v2.01 and have a few questions. My RNAseq data is from 3 time points where each timepoint has a control C1, C2, C3 and an experimental condition E1 , E2 and E3. I have created cuffdiff output for all pairwise comparisons C1_E1, C2_E2, C3_E3. Should I save all cuffdiff output into a single directory for creating plots for all conditions? Also how do you save plots that are generated with CummeRbund?
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Did you run cuffdiff 3 times for each paired comparison? Then you'll loose the biological replicate variance and have to rely on cuffdiff's guessed variance. But seeing as the samples are paired, I don't really know how you would do it otherwise. I'm actually wondering this since I have paired data but don't know how to keep the paired information when running cuffdiff. For example: Family1_sampleA, Family1_sampleB, Family2_sampleA, Family2_sampleB, etc.Originally posted by godzilla07 View Post
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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