Topic
Normalization choice
Which normalization is right for my question, and which ones must never be mixed?
Library-size normalization assumes most genes do not change; that assumption fails for global shifts, cell-type composition changes and spike-in designs. Median-of-ratios, TMM, CPM, TPM and SCTransform answer different questions and are not interchangeable inputs to downstream tools.
- How to Choose a Normalization Method in Spatial Transcriptomics
Library-size normalization removes the exact tissue-architecture signal you're trying to map, and the scRNA-seq default you already know is often the wrong call here.