Hi, This is related to the DESEQ2 Normalization. First I would describe my experiment design, I have two groups, GroupA and GroupB. Group A has 39 replicates (Biological), and Group B Has 77 replicates. The samples are human blood samples, collected and processed randomly for both groups.

RNA-Sequencing was carried out in the 3 batches, However, I have made sure that the samples from both groups should be included in each batch, therefore, I can use the deseq2 design during analysis.

I have run DESEQ2 and run differential expression analysis. I have found around 6700 genes are differentially expressed (padj < 0.05). Around 80 % of genes are downregulated. That was surprising to me. To explore possible reasons I started looking for the normalization factor calculated by the DESEQ2 and when I made a box-plot of the calculated normalization factors for both groups, the normalization factor for Group B (treatment) was significantly different than Group A. I have run the t.test and Wilcox.test and the pValue is 0.00025. I am attaching an image of the box plot for purpose of the illustrations .

Please can you suggest that my analysis is alright or something is wrong?

Thanking you.

dds <- DESeqDataSetFromMatrix(countData = rsem.gene.count.134,colData = design.m,design = ~Batch+class)

dds <- estimateSizeFactors(dds)

factor <- data.frame(factor = dds$sizeFactor, class = dds$class)

library(ggplot2)

p <- ggplot(factor,aes(x = class, y = factor, color = class)) + geom_boxplot() 

p + stat_compare_means(method = "wilcox.test")


# include your problematic code here with any corresponding output 
# please also include the results of running the following in an R session 

sessionInfo( )

Is you sequencing depth confounded with the condition? That would be an issue in the experimental design, not in the analysis. E.g. colSums(counts(dds)) over condition.

If so, I would recommend to do higher count filtering, e.g. require a minimal count (10) in the majority of samples.

keep <- rowSums(counts(dds) >= 10) >= X
dds <- dds[keep,] # before DESeq()