to pool or not to pool

Question

[eyonesi]

Hi all in experimental design for de novo transcription analysis (de novo RNA seq analysis) which method is preferred' to pool or not to pool of replicates? thank you

[st.ph.n]

Pool, to create a de novo transcriptome assembly, if you plan to do downstream DGE analysis between your samples.

[eyonesi]

thank you st.ph.n its OK I'm going to do DGE. but which method is preffered or is better?

[eyonesi]

exactly I asked about pooling extracted RNA from replicates of each treatments to create libraries. So which one is better ' pooling RNA or not pooling? and also if we pool the RNA, isn't it complicate the analysis and assembly in a de novo analysis?

[WouterDeCoster]

Please use ADD REPLY or ADD COMMENT to answer to earlier reactions, as such this thread remains logically structured and easy to follow. I have moved your reaction, but as you can see it's not optimal.

It's better for any type of analysis to keep replicates separately. When you are analysing the data it's no problem to combine the obtained reads (which would mimic pooling) but the other way around is obviously impossible.

[eyonesi]

dear WouterDeCoster thank you

Answer 1

[st.ph.n]

It is both preferred, and better. In order to map the raw reads from the different samples, you should pool your samples by read orientation (all R1's, and all R2's) into the assembly. If you're using Trinity, they specifically recommend this:

If you have multiple RNA-Seq data sets that you want to compare (eg. different tissues sampled from a single organism), be sure to generate a single Trinity assembly and to then run the abundance estimation separately for each of your samples.

[WouterDeCoster]

That's in silico pooling, I think OP is asking about pooling libraries.