Question: DESeq2 vs TCGAbiolinks normalization methods
1
gravatar for array chip
20 months ago by
array chip360
array chip360 wrote:

Hi all, I am still new to RNA-seq analysis. I understand DESeq2 offer rlog() and vst() as a normalization method to raw count data before running downstream analysis. I just came across TCGAbiolinks package for accessing the TCGA datasets. This package also offer its own normalization methods. Here is the description of the normalization options it offers via function TCGAanalyze_Normalization():

TCGAanalyze_Normalization allows users to normalize mRNA transcripts and miRNA using the EDASeq package (28). This function uses within-lane normalization procedures to adjust for gene length or GC-content effects (or other gene-level effects) on read counts: LOESS robust local regression and global-scaling, full-quantile and between-lane normalization procedures to adjust for distributional differences between lanes (e.g. sequencing depth).

How do you think of the different normalization methods offered by these 2 packages? It seems rlog() and vst() from DESeq2 are much simpler than the options offered by TCGAanalyze_Normalization(), but do they provide adequate normalization?

And in general, how do these normalization methods above related to RPKM, FPKM and TPM?

Thank you,

John

rnaseq normalization deseq2 • 478 views
ADD COMMENTlink modified 20 months ago • written 20 months ago by array chip360

Thank you Michael for your comments! If I start with raw counts, do your comments suggest I should run EDASeq's normalization first before using rlog() or vst() from DESeq2?

Thanks again!

ADD REPLYlink written 20 months ago by array chip360
1

Yes you can run vst() after EDASeq, see the vignette. I’d recommend vst() for large datasets.

ADD REPLYlink written 20 months ago by Michael Love25k

Thank you again!

ADD REPLYlink written 20 months ago by array chip360
Answer: DESeq2 vs TCGAbiolinks normalization methods
1
gravatar for Michael Love
20 months ago by
Michael Love25k
United States
Michael Love25k wrote:

rlog and vst are *transformations* and are complementary to EDASeq’s normalization which provides per gene x sample scaling factors. The transformations are substitute for the often used log(x+1) and both will use whatever normalization factors or size factors are present in the DESeqDataSet. 

ADD COMMENTlink written 20 months ago by Michael Love25k
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