Phosphoproteomics time series analysis with splines
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rina ▴ 20
@rina-16738
Last seen 8 days ago
France

Hi all,

I have some phosphoproteomics, time-series data that I would like to analyze. I have four conditions (Control, MutA, MutB, MutA+B) and my aim is to i) Identify which phosphosites are behaving differently across time in the double mutatant (MutA+B) when compare to MutA and MutB. Each condition has been measured across 3 time points (1hr, 8hrs, 24hrs), including the control condition. Each timepoint & condition have 4 replicates.

I was reading the limma user's guide but I am unsure about the following:

  • is the approach using splines (9.6.2) possible given that I only have 3 time points?
  • how to select the correct number of dfs?
  • Should I use duplicateCorrelation since the same replicates were measured throught time?
  • Is the following design matrix correct in order to answer my question?

Code:

X <- ns(as.numeric(data$time, df =3)
Group <- factor(data$Treatment)
design <- model.matrix(~Group*X)
fit <- lmFit(dat.mat, design)
fit <- eBayes(fit)
Limma.results = topTable(fit,coef = 1,number = Inf)
candidate_genes <- Limma.results %>%
  filter(adj.P.Val < 0.01)

Thank you in advance!

limma splines • 173 views
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@gordon-smyth
Last seen 2 hours ago
WEHI, Melbourne, Australia

is the approach using splines (9.6.2) possible given that I only have 3 time points?

Not it is not. Your experiment is covered instead by Section 9.6.1 of the User's Guide.

To elaborate a little, with 3 time points you can only estimate 2 independent contrasts between the time points, so trying to estimate a spline curve with 3 independent parameters is futile. In general, you need to have substantially more time points than spline df.

Should I use duplicateCorrelation since the same replicates were measured throught time?

Yes.

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Hi Gordon! Thank you for your reply.

Do you have any other recommendations instead of splines in order to compare the temporal profiles between conditions and cluster genes that have similar behavior across time?

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Your experiment is covered by Section 9.6.1 of the User's Guide, including how to compare temporal profiles between conditions.

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Thank you, Gordon!

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