technical replicates and normalization/log transformation
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@a-14337
Last seen 12 hours ago
United Kingdom

hi,

I have a question regarding technical replicates of the same biological sample.

I have for a sample the following:

sample1-rep1
sample1-rep2

Sample2-rep1
Sample2-rep2
etc up to 12


The individual samples are biological replicates while the the sample (rep1 and 2) are technical replicates from the same tissue.

My question is, for DESeq2, should this cause issues for normalization/VST transformation? I primarily need the normalize counts/vst transformed counts for further downstream analysis (not DE)

for my model effect, batch is treated according to when library preps took place.

please note, i do not intend to collapse technical replicates.

thanks!

deseq2 normalization • 259 views
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@mikelove
Last seen 1 day ago
United States

We do recommend to collapse the technical replicates if you want to examine or test on the biological differences.

I don't see a problem having the technical replicates in for a VST PCA plot though.

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great! thank you for this advice.

As a follow up, is there a way to generate normalized counts without running the model? For example, would this work?

deObject<- DESeqfrom...etc
deObject<-estimateSizeFactors(deObject)
df<-counts(deObject, normalized = T)

and

vsd <- vst(dds, blind = T)

mat <- assay(vsd)
mat <- limma::removeBatchEffect(mat, vsd\$batch)
assay(vsd) <- mat
write.csv(assay(vsd etc..)


Presumably the model does not need to be run in order to generate normalized counts as above? thank you again

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Yes, normalized=TRUE only requires estimateSizeFactors to be run.

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thank you very much for your time :)