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Old 01-02-2013, 10:47 AM   #1
aimeetal
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Location: Austin, TX

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Default Using Trinity with no replicates

Hello,
For financial reasons my lab could not use replicates for the 2 conditions of my RNA seq experiment... I am a grad student . I performed a differential expression analysis using Trinity with EdgeR (the built-in command).. I did get a subset of differentially expressed contigs. Does anyone know how Trinity/edgeR calls these particular contigs as differentially expressed when there are no replicates? The commands are all pre-set, so I'm not sure what is going on there.
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Old 01-02-2013, 11:37 AM   #2
chadn737
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1) Do everything you can to convince your boss to spend money on replicates

2) Look up the EdgeR package and read about it and what it does

3) Open up the run_EdgeR.pl and try to understand what commands it is feeding EdgeR, it also helps if you post the exact arguments you used.

4) I am not familiar with protocols by Trinity, but it looks like they may be feeding EdgeR RPKM normalized data rather than raw reads. I could be wrong, I did not look at the run_EdgeR.pl too closely. If this is the case, then this is an incorrect way of doing differential expression (not to mention the lack of replicates).
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Old 01-02-2013, 01:11 PM   #3
aimeetal
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haha @ #1.. yes very true. Thank you so much for the advice. I will do that.
I'm wondering if it would almost make more sense to just extract the raw counts from the sam files and calculate the fold changes.. or run the raw counts through RSEM to normalize them somewhat and calculate the fold changes.. I would hate to think the Trinity pipeline uses incorrect commands when I am so trusting of what it is telling me to do
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