I am trying to use
DESeq2 for amplicon data analysis, but I am getting some problem.
Details of the analysis:
here is the summary of data, I do have three tissue: soil, root, and shoot sample collected from different villages.
and we have given three treatment to each soil, root and shoot sample in each village: TREATMENT T1, T2 AND T3.
Now, the aim is to find which microbial species or genus are more abundant in Treatment T1 and Treatment T3 in comparison to T2 and how T3 microbial abundance is different from T1 Treatment.
I tried to pass the phyloseq object into DESeq2
# Phyloseq to Deseq psfdds<-phyloseqtodeseq2(ps0, ~1) # I just pass 1 as design here, because at later step I am going to pass again group as design. #Group Tissue and Tremant variable into one varibale psfdds$group <- factor(paste0(psfdds$Tissue,psfdds$Treatment)) # group used as design again design(psfdds) <- ~ group #Deseq psfdds <- DESeq(psfdds, test="Wald", fitType="parametric") estimating size factors estimating dispersions gene-wise dispersion estimates mean-dispersion relationship final dispersion estimates fitting model and testing -- replacing outliers and refitting for 10886 genes -- DESeq argument 'minReplicatesForReplace' = 7 -- original counts are preserved in counts(dds) estimating dispersions fitting model and testing resultsNames(psfdds)  "Intercept" "group_LeafT2_vs_LeafT1" "group_LeafT3_vs_LeafT1"  "group_RootT1_vs_LeafT1" "group_RootT2_vs_LeafT1" "group_RootT3_vs_LeafT1"  "group_SoilT1_vs_LeafT1" "group_SoilT2_vs_LeafT1" "group_SoilT3_vs_LeafT1" # Now extracting result for soil in Treatment1_vs_Treatment2 res_soil.T1_soil.T2_new <- results(psfdds, contrasts=c("group","TissueSoil.TreatmentT1","TissueSoil.TreatmentT2")) # but when I look at summary of result of any comparison it give same value as shown below summary(ressoil.T1_soil.T2_new) out of 59297 with nonzero total read count adjusted p-value < 0.1 LFC > 0 (up) : 58684, 99% LFC < 0 (down) : 3, 0.0051% outliers  : 0, 0% low counts  : 3004, 5.1% (mean count < 0)
But it seems like there is some problem as it is showing the same result after passing anything.
Could anyone please suggest how can I resolve it.
I will be very thankful to you.