Hi everyone, hope all you are well!
I would like to ask for help and orientation about what to do. I made a DE analysis and there is no difference between the two groups... briefly, the same parasite hosted in two different viscera from different animals but same species , I have 6 samples of every group. I have 6 parasites from sheep lung and 6 parasites from sheep liver. I expected to find differences between the groups but are the same (about 3 genes, hypothetical proteins, with FDR 0.01 and FC 2.0) I expected differences to , for example perform a GO analysis, but now I do not know what to do
So, I really appreciated if you may tell me what more can I do with this result, any other analysis or whatever. If you may show me some paper to read and get ideas , would be really great. I'm really new in this topic...hope you may help me Thanks a lot!!!
Those are relatively stringent cutoffs. Have you tried relaxing them? Maybe your differences just aren't very large.
Hi! Yes i "relaxed" The criteria, (for example 0.05 And 1.5FC) and it was quite similar....thank You!
Are there reference genomes for them, or did you perform a de novo assembly? Did you separate the host and parasite reads? Did you perform the differential expression analysis for the parasite, the sheep, or both?
Hello! Sorry for not being si clear. I have a reference genome And i only have parasite reads (i do not have víscera reads) . The aim is to observate DE of The sale parasite un different víscera, so i'm only looking at The parasite genes. Thank You!
What you're comparing is not entirely clear. Is it infected liver to infected lung? Do you detect any difference between uninfected liver and uninfected lung and between uninfected organ and infected one? Review your experimental design, data quality and data processing steps. Assuming no problems are found, you may want to consider the conclusion that there is no difference however disappointing this may be. It might be worth redoing the experiment including some form of positive control to ensure that if there's a difference you can detect it.
Thank you for your answer! In The previous answer i tried to write it again to be clear Thank You!
Is there any reason to think that the parasite would behave differently in terms of gene expression based on the tissue it's in? If there is, I would redo the experiment such that you can be confident you're not missing a difference if it exists. If there's no reason to think there's a difference then I'd take your result as preliminary evidence there's no difference. In this case you have to decide whether it's worth redoing the experiment with appropriate controls to confirm this result.