User: Frederik Ziebell

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Posts by Frederik Ziebell

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Using RUVs with known batches
... I have a large (bulk) RNA-seq data set with ~1500 samples, i.e. ~30 multiplexing runs (library prep and sequencing) with in total ~500 different conditions in triplicates (conditions are somehow randomized across runs). The ultimate goal is to test all 500 conditions against the wildtype-controls (w ...
deseq2 ruvseq written 11 weeks ago by Frederik Ziebell0 • updated 10 weeks ago by Michael Love25k
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Comment: C: lmFit very slow if there are missing values
... Thank you for the clarification. The actual dataset I have contains many conditions and so lmFit takes about half an hour, that's why I initially viewed it as a bug. Just out of curiosity, what's the super-fast algorithm that only works if there are no NAs? ...
written 11 weeks ago by Frederik Ziebell0
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lmFit very slow if there are missing values
... Having only a single missing value slows lmFit down by over an order of magnitude: library("limma") library("tictoc") n_genes <- 10^6 sd <- 0.3*sqrt(4/rchisq(n_genes,df=4)) y <- matrix(rnorm(n_genes*6,sd=sd),n_genes,6) y[1:2,4:6] <- y[1:2,4:6] + 2 d ...
limma written 11 weeks ago by Frederik Ziebell0 • updated 11 weeks ago by Gordon Smyth39k
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Comment: C: DESeq2: Poor dispersion fit, even when a local or custom fit is used
... Hi Mike, thank you for having a look at my data! I'm also aware of the problem of "almost" confounding, but thought I could get away with it, since most runs share two conditions with other runs. We are currently setting up new runs to re-measure previous conditions and de-confound the design. How ...
written 17 months ago by Frederik Ziebell0
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Comment: C: DESeq2: Poor dispersion fit, even when a local or custom fit is used
... Here is the plot when setting n to the samples in a condition, which makes up a biological group: Even when I use a more aggressive filter and set n to the number of samples in a run (48), the problem still remains: ...
written 17 months ago by Frederik Ziebell0
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Comment: C: DESeq2: Poor dispersion fit, even when a local or custom fit is used
... Hi Mike, I tried several filtering methods but none seemed to resove the problem. I made the problem smaller by just keeping two genes in the data with similar baseMean and variance. If I use ~condition+run as design, one gene gets a very low dispersion estimate and the other a very high one (upper ...
written 17 months ago by Frederik Ziebell0
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Comment: C: DESeq2: Poor dispersion fit, even when a local or custom fit is used
... Hi Mike, I followed up with your suggestion and figured out the source of the batch effect. It was a different read-length that affected the mapping, since I was using STAR with splice junction overhang equal to read length minus 1. After trimming all reads to the same length, the effect is gone: ...
written 17 months ago by Frederik Ziebell0
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Comment: A: DESeq2: Poor dispersion fit, even when a local or custom fit is used
... Hi Mike, indeed there are batch effects in the data. The way samples are processed is that 48 samples are always multiplexed together. So for these samples, library prep is performed, followed by sequencing and the combination of both is encoded as a "run". Here is a PCA of the vst-transformed coun ...
written 18 months ago by Frederik Ziebell0
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DESeq2: Poor dispersion fit, even when a local or custom fit is used
... I have a RNASeq data set with ~650 samples. When I run DESeq2, I get a very poor fit of the mean-dispersion trend: Here is also a plot of the original trend (without final dispersion estimates), and color coded the number of dispersion iterations: This is the output of DESeq: ​In DESeqDataSe ...
deseq2 dispersion written 18 months ago by Frederik Ziebell0
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Comment: A: rlog transformation producing outliers with very high log-transformed counts
... Hi Mike, thank you for your explanations. The reason I like to use rlog() over vst() is that it achieves almost equal within-sample distributions of counts across all samples, which may be connected to the size factor issue you were mentioning. Do you see potential future improvements of rlog() usi ...
written 21 months ago by Frederik Ziebell0

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