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Thank you so much, @lgeistlinger !
Here are the updates:
design = ~ batch + condition worked!
(Results were not very different with or without age, thanks to you, age is no more covariate of interest in my analysis.)
I could not proceed without normalization:
res <- sbea(method = "gsea", se = se, gs = go.bp.gs, browse = T)
Error in .reorderAssays(se, assay) :
Expression dataset (se) does not contain an assay named "norm"
Normalization worked but excluded ~91 % of genes:
se <- normalize(se, norm.method = "vst")
Excluding 8118 genes not satisfying min.cpm threshold
The results were from DESeq2 and not from raw read counts. Considering the vignette, I am not sure if normalization is needed. Also, DESeq2 output already has normalization factors:
How can I make use of DESeq2's normalization factors to normalize the count slot and create "norm" slot in an SE?
(I think, it's an important consideration for the import() function.)
> se <- import(dds, res)
> se
class: RangedSummarizedExperiment
dim: 64102 8
metadata(4): '' version design dataType
**assays(2): raw norm**
rownames(64102): ENSG00000000003 ENSG00000000005 ... LRG_98 LRG_99
rowData names(27): baseMean FC ... deviance maxCooks
colnames(8): SRR1039508 SRR1039509 ... SRR1039520 SRR1039521
colData names(11): SampleName cell ... sizeFactor GROUP
will automatically attach an additionally assay norm that contains the variance-stabilized expression values that are normalized for differences in library size between samples.
The resulting se can then be used to carry out enrichment methods such as gsea:
> res <- sbea("gsea", se, kegg.gs)
Permutations: 1 -- 100
Processing ...
Thank you so much, @lgeistlinger !
Here are the updates:
design = ~ batch + condition
worked!(Results were not very different with or without age, thanks to you, age is no more covariate of interest in my analysis.)
I could not proceed without normalization:
Normalization worked but excluded ~91 % of genes:
The results were from DESeq2 and not from raw read counts. Considering the vignette, I am not sure if normalization is needed. Also, DESeq2 output already has normalization factors:
How can I make use of DESeq2's normalization factors to normalize the count slot and create "norm" slot in an SE?
(I think, it's an important consideration for the
import()
function.)Originally posted by @vivek-verma202 in #23 (comment)
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