Hello

Thank you for developing this great tool. I have a very basic question.

I want to use makeExampleDESeqDataSet to simulate RNA-seq data using pilot data, to test the power of different sample sizes in an experiment with two treatments.

However, I am confused about how to input the parameters from my pilot data. Disclaimer - I am a biologist not a statistician. I have tried to understand the methods section of the DEseq2 paper, but unfortunately I still don't follow how to do this.

For example, from my pilot dataset I have the mean expression and mean dispersion; how do these relate to the input parameters of makeExampleDESeqDataSet? ie betaSD /interceptMean / dispMeanRel / sizeFactors. I plan to simulate the treatments separately at different sample sizes, and then calculate differential expression and cohen's D for each.

Thanks

Lily

dispMeanRel would be dispersionFunction(dds) from a pilot dataset. It's the relationship between dispersion and mean.

betaSD is not known, you have to provide this. It's the SD of the true LFC. Maybe 1? Again you have to provide this and motivate it for your dataset.

interceptMean is the log2 of the count. So if you plan to have depth of 1000 for most genes, that be specified with interceptMean = 10. Likewise interceptSD specifies how the depth varies across genes. If you put 0 then all genes have the same depth.

sizeFactors can be left at default, unless you plan to have differential sequencing depth across samples (not advisable).