I am currently using deseq2 package to normalize my data and after that I will use it to calculate p-values between my conditions. I have a question about after normalization step. I normalized my RNA-seq data and I used this normalized data to find outliers in my data, I used PCA analysis. There are some samples that will be removed. But when checked the code I see that normalization step is inside the deseq function and I can not replace my new data (the data which is normalized previously but outliers removed) to the deseq function. Obviously it seems that, I will remove the outliers and renormalized my data to calculate p-values.
when I check some manuals I saw this:
"NOTE: DESeq2 doesn’t actually use normalized counts, rather it uses the raw counts and models the normalization inside the Generalized Linear Model (GLM). These normalized counts will be useful for downstream visualization of results, but cannot be used as input to DESeq2 or any other tools that peform differential expression analysis which use the negative binomial model." in here: https://hbctraining.github.io/DGE_workshop/lessons/02_DGE_count_normalization.html
I want to be sure that there is anything I can do to replace my data into deseq object. If there is a way to do it, can you explain to me please?