using tcga and gtex to look for lncrna DE (using raw files) - what are the best ways to normalize? deseq2 and edger? also if i want to look at lncrnas of specific chromosomes, how should i approach normalization?
1 answer
The real answer to this is that you can't. TCGA and GTEx are different experiments and they aren't really comparable in a rigorous way on a quantitative level. At best you might find leads that would need extensive lab validation. No reviewer who knows what they are doing will accept a paper based solely on a DE comparison between TCGA and GTEx.
If you were insistant on doing this then I can see two appraoches:
- Most TCGA datasets have at least some normal controls. Set up a design table with two columns: Disease (fill with Cancer and normal) and Dataset (fill with TCGA and GTEx). Set up your design formula to be '~ Dataset + Disease'. EdgeR or DESeq2 doesn't matter, they are more or less the same. Note this will only work if your TCGA data has at least some normals in it.
- Transform the data with limma voom. Then apply limma's quantile betweenArrayNormalization. This is a super harsh normalization.
But just to reiterate - I wouldn't trust the results that came out of either of these. I'd treat this as lead prioritisation only.
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