Question

Clarification requested for the RNAseq Gene workflow document

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lina.faller • 0

@linafaller-9082

Last seen 7.0 years ago

Hi all,

I am looking at the following bioconductor document: http://www.bioconductor.org/help/workflows/rnaseqGene/#time-course-experiments

Towards the end of the documents, there is a heatmap with the following title: "Heatmap of log2 fold changes for genes with smallest adjusted p value"

It references the log2FC values of the comparisons performed in the following list:

"Intercept"
"strain_mut_vs_wt"
"minute_15_vs_0"
"minute_30_vs_0"
"minute_60_vs_0"
"minute_120_vs_0"
"minute_180_vs_0"
"strainmut.minute15"
"strainmut.minute30"
"strainmut.minute60"
"strainmut.minute120"
"strainmut.minute180"

I assume that (2) are the log2FC from the comparison of mutant to wildtype strain (at time = 0?) and (3) is the comparison of two timepoints, 15 minutes vs baseline at time zero, taking into account the effect of the strain.

However, what are the comparisons performed for (8)-(12)?

Is (8) wildtype strain vs mutant at 15 minutes? Or is it the mutant strain at 15 minutes vs 0 minutes?

Thanks for any insight!

deseq2 rnaseq • 1.3k views

ADD COMMENT • link updated 7.0 years ago by Michael Love 41k • written 7.0 years ago by lina.faller • 0

score 1 · Answer 1 · 2017-05-11

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Michael Love 41k

@mikelove

Last seen 17 hours ago

United States

These are interaction terms. Take a look at this section of the DESeq2 vignette:

http://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#interactions

ADD COMMENT • link 7.0 years ago Michael Love 41k

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Great, thank you for the reference!

The interaction terms genotypeII.conditionB and genotypeIII.conditionB give the difference between the condition effect for a given genotype and the condition effect for the reference genotype.

So the "strainmut.minute15" column shows the log2FC for the mutant at 15 minutes compared to the wildtype at 15 minutes?

In that case, there does not seem to be a column for mutant at 0 minutes vs wildtype at 0 minutes in the heatmap. However, the betas variable does contain a column labeled "strain_mut_vs_wt" which was removed before plotting the heatmap -- is that the data at time point zero?

ADD REPLY • link 7.0 years ago lina.faller • 0

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Yes, the main effect strain_mut_vs_wt is the mutant vs WT effect at time 0, and the Intercept is then the WT base-level expression at time 0.

ADD REPLY • link 7.0 years ago Michael Love 41k

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Great! And if a gene has a positive log2FC in the "strain_mut_vs_wt" column, then it is upregulated in the mutant strain (because the mutant strain is listed first in the description")?

ADD REPLY • link 7.0 years ago lina.faller • 0

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Yes, you can read more about how to interpret log2 fold changes in this section of the workflow:

http://www.bioconductor.org/help/workflows/rnaseqGene/#differential-expression-analysis

"The column log2FoldChange is the effect size estimate. It tells us how much the gene’s expression seems to have changed due to treatment with dexamethasone in comparison to untreated samples. This value is reported on a logarithmic scale to base 2: for example, a log2 fold change of 1.5 means that the gene’s expression is increased by a multiplicative factor of 21.5 ≈ 2.82."