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I am trying to learn how to analyse normalised DIA-MS data and I am struggling with it ://

The original dataset I got is (6 conditions (2 samples each)) with 3 technical replicates (total: 36 sample columns x 1128 proteins in rows). I removed the proteins with more than 80% of NA, and impute with kNN the rest of missing values.

Then I collapse the replicates doing the average of the 3 replicates (is this correct?)

So now, I have a dataframe with 12 columns x 1072 rows.

The question is... I am not sure how to perform the differential analysis from here (assuming I did well the previous steps lol)

Could someone help me with the tools or code to compare all the 6 conditions between them?

I read something about Limma package, but not sure if it can be used in this DIA-ms data and neither sure of how to use limma for it (I already check the vignette with no succeed)

UPDATE: I tried to use Limma as follows:

fit1 <- lmFit(norm.data_collapsed, design)
fit2 <- contrasts.fit(fit1, contrasts = contrast)
fit3 <- eBayes(fit2)
limma.res.01 <- topTable(fit3, coef = comb.contrast[1], n= Inf)

This returns me a non-sense values for logFC or significance:

head of the results topTable

Any help would be very welcome,

Best regards

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Regarding the big FC values, I just realise the Limma lmfit function requires as input a log2 matrix... so I just transformed my data and now makes more sense. Still I have the doubt regarding the collapsing technical replicates and and if some one can verify the pipeline would be like that.

Thanks!!!

Fer

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