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6.0 years ago
DVA
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Is the "-M" option necessary when using Cufflinks? I understand it would prevent downstream analysis focusing on regions like rRNA, but could this be done at the very end after differential expression? Also, I could not find a mask gtf to download from USCS genome browser... Any advice please? Thank you.
You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.
Thanks a lot. I was trying to repeat a previous analysis done by a colleague.
Alright, makes sense, but probably also interesting to see if you can replicate the findings with a more recent method.
Yes absolutely. That is what I will do next. Thank you!
Do you know if my library is 3' rna seq library, what parameters should I alter with StringTie? Ideally, for 3' rna seq lib, transcript assembly is not needed for our DE analysis. Some default setting such as 200bps in -m, minimum length allowed for predicted transcripts，will be not relevant.
What I have in mind is the alternative workflow on : http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual (second workflow under "Differential Expression Analysis).
So you don't have full RNA-seq? Then I doubt whether stringtie is going to be much use, there is not a lot to assemble. I would suggest you use featureCounts or salmon for quantification and go directly to DESeq2 or edgeR.