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4.9 years ago

evlampia123x
•
0

Hello!!

I did an RNA seq and then I did the normalization of TMM. Afterwards I chose:

- 1 contig which was over-expressed
- my 4 plants populations
- the TMM values regarding this specific contig and the 4 populations.

So I thought to do an ANOVA test to check for any st.sign. diff. among the populations for the specific contig. In the end I did a t test with Bonferroni adjustment.

Do you think that this whole thing I did make sense? If not what you could recommend to me?

Btw I tried the heatmap but I think my excel set up is somehow wrong...

Thank you very much in advance!

Any reason why you did not use an algorithm such as DESeq2 or edgeR for your analysis?

I did. I used edgeR...

Then why the ANOVA test? I'm afraid I don't understand your question.

Sorry. It s my fault. I am beginner in Bioinformatics. I did it with edgeR. And then I did an exact test analysis in Blast2GoPro based on some threshold for logFC, FDR, p value. And I took the contigs with FDR<0.05 as upregulated. Now I want to demostrate them as a figure. So now I can plot the TMM as a boxplot for my different populations and write on the top of my plot the FDR value. But I want also to show

statistically significant differences btw the populations.The FDR is only about the contig. I dont have a unique FDR for each population. So my question is: How to show st.sign.differences btw the populations regarding the same contig? Sorry for the confusion....I think the question was why didn't you go ahead and use edgeR/limma for the statistics part? Most people will use the linear modeling in limma/DESeq2 for statistical testing.

I dont know what are these stuff...sorry...

Hi evlampia123x,

there are a few things that need clarification: What exactly do you mean by contig? Is contig = gene? So far, I understand that you did RNA-seq but which groups did you compare with edgeR? Do you have 4 populations under different conditions or compare 2vs2? What is the experimental setup? Please add some details to help understand your problem.

I did RNA sequence using untreated plants coming from 4 different populations. I had 6 plants from each population. So overall 24 untreated plants. Then I took the results of the sequence and aligned them against a reference transcriptome. Then I did an BWA-MEM mapping. Then I took the raw data results and then I took the TMM (normalization) values (all these took place online in GeneData). When I say contig I mean contig which corresponds to a gene (let s say contig=gene). And I compared everything with edgeR (GeneData). Like this I took the TMM values. Are these infos enough? Sorry for the confusion. I am a beginner....if you cannot help me I will understand it...it s my fault bcs I cannot give you enough details because I dont know many things. I did the analysis with an other person...My question actually is: Is it

correctto anANOVAfor TMM values or this is absolutely bulshit? Thank you guys for your help!!