Notes from the bench
Specific troubleshooting, tool comparisons, and workflow notes from real client projects. No "intro to RNA-seq" — only the parts that actually trip people up.
Over-representation analysis throws away every gene below your cutoff. GSEA ranks all of them. When that difference decides whether you see the biology, and the two settings that make or break the result.
The 2-fold cutoff is a cell-line habit that quietly discards real signal in clinical RNA-seq. When to lower it, why lfcShrink matters, and how to test against a threshold instead of filtering after the fact.
End-to-end RNA-seq workflow: QC, alignment, quantification, DESeq2, pathway analysis, reporting. The five stages, the real tool choices, and what production looks like in Snakemake.
Running PCA before differential expression takes 30 seconds and catches batch effects, mislabeled samples, and outliers that silently destroy DESeq2 results. How to read it.
Why tumor-only somatic calling is harder than the docs admit, and a layered filter strategy that keeps false-positive rate manageable: PoN, gnomAD AF, and signature-based germline cleanup.