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Question: Data normalization and transformation using DESeq2
0
gravatar for chiara.facciotto
11 weeks ago by
chiara.facciotto0 wrote:

Hi,

I am trying to use DESeq2 to perform normalization of my RNA-Seq data. I am wondering if, after estimating the size factors, I can directly run the rlog or vst transformation, or if I also need to use the count function in between. Basically I wonder which is the correct one between option 1 or option 2 of the codes reported below (in bold I highlighted the difference between the two scripts).

Thank you very much for your help!!!

Option 1:

  # Import data
  dds <- DESeqDataSetFromMatrix(countData = counts, colData = colData, design = ~ 1)

  # Pre-filtering
  dds <- dds[ rowSums(counts(dds)) > 0, ]

  # Estimate factor for normalization
  dds <- estimateSizeFactors(dds)

  # Compute log2 counts
  rld <- rlog(dds, blind=FALSE)
  table.out <- assay(rld)

Option 2:

  # Import data
  dds <- DESeqDataSetFromMatrix(countData = counts, colData = colData, design = ~ 1)

  # Pre-filtering
  dds <- dds[ rowSums(counts(dds)) > 0, ]

  # Estimate factor for normalization
  dds <- estimateSizeFactors(dds)
  dds <- counts(dds, normalized=TRUE)

  # Compute log2 counts
  rld <- rlog(dds, blind=FALSE)
  table.out <- assay(rld)
ADD COMMENTlink modified 11 weeks ago • written 11 weeks ago by chiara.facciotto0
Answer: Data normalization and transformation using DESeq2
0
gravatar for Michael Love
11 weeks ago by
Michael Love22k
United States
Michael Love22k wrote:

The counts() function doesn't do any normalization, it just returns a matrix. So you don't want to do this: dds <- counts(dds, ...), because you've just replaced a DESeqDataSet (which has a lot of information in it) with a count matrix (which has less information). You've discarded all the information about genes and samples.

ADD COMMENTlink written 11 weeks ago by Michael Love22k
Answer: Data normalization and transformation using DESeq2
0
gravatar for chiara.facciotto
11 weeks ago by
chiara.facciotto0 wrote:

Thanks! And is it ok to runĀ estimateSizeFactors(dss) before running rlog(dds, blind=FALSE)?

ADD COMMENTlink written 11 weeks ago by chiara.facciotto0

Yes, you should. But if you haven't done that already, the rlog function will do that internally.

ADD REPLYlink modified 11 weeks ago • written 11 weeks ago by Steve Lianoglou12k
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