Question: Configuring sleuth for log2FC - What are the dangers?
2
gravatar for Lorenz Becker
10 months ago by
Manchester, UK
Lorenz Becker20 wrote:

(Detailed) Background:

I'm working with Prof. Love's kallisto/sleuth and DESeq2 libraries, on my PhD, where I run an RNA-seq timecourse experiment comprising 6-8 samples per timepoint over four (unevenly spaced) developmental timepoints.

My current model is ~ batch + CutRIN + ns( ImputedAgeDays) - that is, if I understand correctly, it corrects for batch effects and accounts for low/medium/high RIN (cut so as to avoid using a numeric factor, which DESeq2 models in a specific way; my apologies, Prof. Love, I know you discourage the inclusion of RIN in DESeq2), then fits a spline to the (estimated) age of the sample in days (which means the fit is smoothed better than processing counts without a spline would be).

Running an LRT, with reduced= ~batch + CutRin, (again, if I understand correctly) I get a list of genes whose expression over time fits the spline better than noise (i.e. change over time), with sleuth outputting more than DESeq2.

Question:

sleuth log-transforms data before fitting, and outputs beta, an effect size, which is not the same as a fold-change or log fold change. Therefore, sleuth effect sizes cannot directly be compared to DESeq2 output and its log2FCs. By calling sleuth_prep() with transformation_function=log(x+0.5, 2), sleuth can output log2 fold changes. However, there is a warning, be sure you know what you're doing before you change this.

What is that warning about?

There must be a good reason, but I've not found an explanation anywhere, and whilst I can assume there are assumptions e.g. in the way sleuth models the data and bootstraps, I am not familiar enough with the matter to see which might be violated. What dangers are there if I switch sleuth to log2FCs, and would I be better off just ranking the results by beta/log2FC to compare sleuth results to DESeq2?

rna-seq sleuth • 876 views
ADD COMMENTlink written 10 months ago by Lorenz Becker20
1

It is not clear from your question if you are analyzing over genes for both DESeq2 and sleuth, or genes for DESeq and transcripts for sleuth, or transcripts for both.

Maybe the kallisto-sleuth-users is a better place to ask this question, there have been related discussions before - here, here, here and here .

ADD REPLYlink written 10 months ago by h.mon22k

Thank you for the referral!

ADD REPLYlink written 10 months ago by Lorenz Becker20
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