Hi everybody, I used Hisat2, Stringtie, and ballgown for my RNAseq analysis. I have 173 samples coming from around 60 birds with 3 different tissues: testis, tectum (in the brain), and pallium (in the brain). and we did also an experiment on the birds and we have two groups: single-pair and double-pair. I tried to do the differential gene expression with Ballgown for all of the 173 samples in a single analysis because I need to compare the differential gene expression between different tissues specially compare two different brain areas together. the first part of my pheno_data look like this now:
![Pheno_data] https://ibb.co/61zCfRs ID, Ring No., Tissue, Treatment, first samples: 10pall_bk792 ,bk792 ,pallium ,Single-pair second sample: 10tec_bk792 ,bk792 ,tectum ,Single-pair third sample :11pall_bk796 ,bk796 ,pallium ,Double-pair forth sample: 11tec_bk796 ,bk796 ,tectum ,Double-pair fifth sample:12pall_bk797 ,bk797 ,pallium ,Double-pair ......................
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I used this command however I am not sure is correct.
results_transcripts= stattest(bg_filter, feature="transcript", covariate = "Treatment", adjustvars=c("Tissue"),getFC=FALSE, meas="FPKM")
I mean that the p-value that we have in the output is showing the difference between treatment considering the tissues? Since we have from the same individual, 3 different samples, we should do something to control for that? Which analysis do you recommend me to do to show variation and differences among two different treatment ?
Can you comment on what I asked in this previous thread since it is relevant here: C: "fold change" question when using ballgown