Hello all,

I got my sequence reads table with FeautureCount and now I am doing my gene expression analysis. I have 18 samples with three groups (one groups received probiotics, one group received antibiotics, and one group as a control, and each have 3 samples, then I replicate this explements). But one of the samples was not good so I end up with 6 samples for controls, 5 samples for Antibiotics, and 6 samples for probiotic groups. These samples are fish samples and all my samples are coming from one family. So basically, all 6 samples for each tank are biological replicate except they are from different tanks (tanks 1 three samples, tanks 2 three samples). First I do not know how to combine my biological replicates. Secondly when I did my sequence analysis, I got only few genes as significant, but because I tested many genes (50/000) genes, I think after correcting for P value, I am getting type two error (falsely rejecting because testing many times). Any help or suggestion? I also attached my meta data and below are my commands. please let me know if my design does not make sense to you.

library(DESeq2)
cts<-read.table("counts1.txt",header=T) 

library(ape)

coldata<-read.table("metadata1.txt",header=T)



dds <- DESeqDataSetFromMatrix(countData = cts,
                              colData = coldata,
                              design <- ~ Treatments)


dds <- DESeq(dds)


resultsNames(dds)
![enter image description here][1]


Treatments_C_vs_Anti <- results(dds, contrast=c("Treatments","![enter image description here][1]C","Anti"))

Treatments_C_vs_Pro <- results(dds, contrast=c("Treatments","C","Pro"))

Treatments_Pro_vs_Anti <- results(dds, contrast=c("Treatments","Anti","Pro"))




resOrderedTreatments_C_vs_Anti <- Treatments_C_vs_Anti[order(Treatments_C_vs_Anti$pvalue),]

resOrderedTreatments_C_vs_Pro <- Treatments_C_vs_Pro[order(Treatments_C_vs_Pro$pvalue),]

resOrderedTreatments_Pro_vs_Anti <- Treatments_Pro_vs_Anti[order(Treatments_Pro_vs_Anti$pvalue),]






write.csv(as.data.frame(resOrderedTreatments_C_vs_Anti), 
          file="resOrderedTreatments_C_vs_Anti.csv")






Here is also how my meta data looks like:

sample_Id   Treatments  TreatmentswithBiologicalreplicate   Tank
J12A.sam    C   C   1
J12B.sam    C   C   1
J12C.sam    C   C   1
J35A.sam    C   C_R 2
J35B.sam    C   C_R 2
J35C.sam    C   C_R 2
J67A.sam    Anti    Anti    1
J67B.sam    Anti    Anti    1
J67C.sam    Anti    Anti    1
J91A.sam    Anti    Anti_R  2
J91B.sam    Anti    Anti_R  2
J115A.sam   Pro Pro 1
J115B.sam   Pro Pro 1
J115C.sam   Pro Pro 1
J139A.sam   Pro Pro_R   2
J139B.sam   Pro Pro_R   2
J139C.sam   Pro Pro_R   2




sessionInfo( )

Unfortunately, my time on the support site has to be limited to specific software questions, and I don't have time to review e.g. analysis plans, or look over people's code.