Differential expression genes
Hello everyone, i was wondering if i working on species don’t have references on NCBI and i want to do Differential expression of this organism in two conditions normal and diseased. If i made first transcriptome reference for this organism, what tools is good for annotation? Second if i need to get differential expressed genes form that data not transcripts level, what tools can help me to do that?
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Depends on whether you are working with a prokaryote or a Eukaryote. You could use
FINDER(LINK) for eukaryotes. When you assemble the transcripts include all data (from all conditions) to build a comprehensive representation of the transcriptome. You can usesalmonorkallistofor the DE analysis once you have the transcriptome assembled.Are you working with something obscure? At this point there should be something available in NCBI, at least a close relative.
So kallisto will do DE on transcript level ?or gene level? Because I need on gene level and another question using RNA seq or mRNA seq will be better?
Yes kallisto and salmon can get you gene-level — you get your transcript quantifications first and then aggregate them to the gene-level.
Not sure what you mean by mRNA-seq… if you mean just capturing the polyA transcripts, then a lot of RNAseq protocols already do that.
mRNAseq (capture of polyA-RNA or ribodepletion) would enrich transcripts compared to total RNAseq (which will also sequence rRNA, ~90% of cellular RNA).
Choice of the method may depend on the amount (~10x ideally needed for mRNAseq), quality (good RIN) of RNA. If you need non-coding RNA then totalRNAseq would be needed.
Try trinityrnaseq.