Question: Estimatedispersions In Deseq
0
gravatar for c9r1y
6.1 years ago by
c9r1y10
United States
c9r1y10 wrote:

I am running DESeq on RNAseq data. I have two conditions, three replicates for each.

I would like to find the top 100 differentially expressed genes and look at them in a heat map.

I have done estimateDispersions with the default settings and then did nbinomTest on the resulting data. I have a list of genes with significant p-values I was thinking were my genes of interest. However, when I look at the DESeq vignette I see that to look at the data on the heatmap, they used estimateDispersions method=blind.

My question is, should I be running nbinomTest with data from estimateDispersions method pooled or blind or another option? Is it fair to use the default settings, choose my top 100 genes, and THEN get variance stabilized data and look at the genes in the heat map?

Thanks for any insight you may have.

ADD COMMENTlink modified 6.1 years ago by Devon Ryan92k • written 6.1 years ago by c9r1y10
1
gravatar for Devon Ryan
6.1 years ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

You need to use the pooled dispersion for the actual DE testing. The blind dispersion method is just use for the variance stabilizing transformation (useful for the heatmap you want).

ADD COMMENTlink written 6.1 years ago by Devon Ryan92k

Thanks for your answer. I've used both for DE testing and I seem to get different statistically significant genes between the methods. Is it reasonable to choose my genes, then do the blind dispersion, VST and heatmap? Or should the blind dispersion method determine which genes I'm calling of interest?

ADD REPLYlink written 6.1 years ago by c9r1y10

Definitely use pool dispersion for the actual DE testing, you'll get more reliable results that way. In the grand scheme of things, you could use either pooled or blind dispersions for the VST. You'll just get somewhat different results in the heatmap since the variance for those genes will be a bit different. The actual look of the heatmap will likely be pretty similar though.

ADD REPLYlink written 6.1 years ago by Devon Ryan92k

Thanks so much, this makes sense they do look fairly similar.

ADD REPLYlink written 6.0 years ago by c9r1y10

Also, another follow up question, is the "blind" method designed mostly for experiments in which there are not replicates? Or what is its purpose?

ADD REPLYlink written 6.0 years ago by c9r1y10

Yup, mostly for experiments lacking replicates and also for heatmaps and the like.

ADD REPLYlink written 6.0 years ago by Devon Ryan92k
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