Mask gtf file for cufflinks
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.
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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.