Differential expression genes

Question

[m90]

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?

[GenoMax]

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 use salmon or kallisto for the DE analysis once you have the transcriptome assembled.

i working on species don’t have references on NCBI

Are you working with something obscure? At this point there should be something available in NCBI, at least a close relative.

[m90]

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?

[dsull]

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.

[GenoMax]

RNA seq or mRNA seq will be better?

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.

[Arup Ghosh]

Try trinityrnaseq.