Hi. there are two issues that I have.
Raw read count used as input data for deseq(). Is it really okay? I referenced that information on following website.http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#why-un-normalized-counts But, I want to try normalizing raw count into RLE(Relative Log Expression).
What does default statistical method of DEseq()? nbinomialwaldtest?
thank you for your help.
# setup condition <- factor(c(c,c,c,t,t,t)) coldata <- data.frame(row.names = colnames(Counts),condition) dds <- DESeqDataSetFromMatrix(countData = Counts, colData = coldata, design = ~condition) # Estimate size factors using Relative Log Expression method dds <- estimateSizeFactors(dds, type = c("ratio")) <------------- I thought this ratio type is about RLE normalization. Is that right? dds <- estimateDispersions(dds) dds <- nbinomWaldTest(dds) <--------- Is this same with just "DESeq(dds)"? # Retrieve normalized counts normcounts <- counts(dds, normalized=TRUE) <----------- How can I apply this to dds? dds$countData <- normcounts? dds <- DESeq(dds) res <- results(dds,contrast = c("condition",t,c))
Thank you for answering my repeated question. Although the documentation states that normalization is not necessary, I was confused because some other parts of the documentation suggested normalization methods.
In fact, I outsourced the RNA sequencing analysis. However, there was a difference between the results analyzed by the company and the results analyzed by me, so I inquired about it. The company does not provide specific information on the grounds of security. The functions used by the company are DESeqDataSetFromMatrix and estimateSizeFactors for RLE normalization, followed by estimateDispersions and nbinomWaldTest.
Therefore, the differences between the results analyzed by me and the company's analysis method are as follows:
I think the difference is due to normalization. When using the above functions, I did not use any special options. However, I do not know what options the company used. Have you had a similar experience?
Either the company provides you full code and software versions or (imo) their anslysis is basically useless as not reproducible.