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