Hey guys,
I just started using Cufflinks on Galaxy (2.2.1) to analysis my RNA-seq data. But the tutorial example I found was quite simple (2 samples comparison) and I got a bit confused. I have the data from 3 different niches and within each niche there were 3 replicates. I did the de novo assembly using transABbyss as there were no reference genome.
I named my samples as follows
SCC1 SCC2 SCC3
SAC1 SAC2 SAC3
SDC1 SDC2 SDC3
I mapped the pair-end reads of each sample against the de novo assembly. Then I was not sure if i should use the data from each sample to do the cufflinks assembly or combine the data from the same niche then do the cufflinks assembly (but i combined the data within same niche anyway).
Next when it came to cuffmerge, I merged all the data across different niches. Later, I did cuffquant analysis using the output of cuffmerge and when it asked for replicates 1, 2,3, I input the datasets from same niche as replicates 1,2,3, respectively. Now I have 3 cbx data file corresponding to each niche (I think so).
Then from the output of the cutdiff, I found there were sample 1(niche 1) and sample 2 (niche 2), but no sample 3 column. Seems the workflow only worked on two-sample comparison
Any idea is welcome.
Thanks
If everything is set up correctly, the second pairwise comparison will be below first in the cuffdiff output, not in another column. So try scrolling down, you should see the names in the sample_1 and sample_2 columns change.
Dear Fang Liu
Here is a reference article.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334321/
Pl look at Figure 2 for the pipeline.
Use CummeRbund to analyse cuffdiff results further.
http://compbio.mit.edu/cummeRbund/.
Hope this will help you.
Best!!
https://pods.iplantcollaborative.org/wiki/display/Events/RNA-Seq+with+CummeRbund
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