Question: RNAseq uneven PCR bias "how to take into best take into account uneven PCR bias ?"
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gravatar for biostar.anon
2.4 years ago by
biostar.anon0 wrote:

Hello, I am a student doing a project with some conceptual difficulties (I do realise such question optimally shouldn't occur). The project aims to compare 8 RNA-seq samples (4 vs 4) testing for differentially expressed genes. The problem is that 2 of the samples (1 for each categories) weren’t amplified as much as the others (an additional 5 cycles).

Obviously, such treatment is sub-optimal for comparative studies, I was wondering what was the best remedy for this problem short of resequencing. I was considering DESEQ2 for it’s outlier capacities or dropping the samples (doing a 3 vs 3) Additional information: the transcriptome is de novo using the samples.

rna-seq • 798 views
ADD COMMENTlink modified 2.3 years ago by Biostar ♦♦ 20 • written 2.4 years ago by biostar.anon0
1

I think that if you remove PCR duplicates and use DESeq2 you shouldn't have a bias

ADD REPLYlink written 2.4 years ago by Asaf7.0k
3

The user then runs the risk of removing actual signal, If you can unequivocally see on a PCA that those two samples are very different from their respective groups, I'd maybe include it as a batch effect in the DESeq2 model

ADD REPLYlink written 2.4 years ago by James Ashmore2.8k
2

Of course you would have a bias! Removing duplicates would incorrectly decrease the inferred expression level of highly expressed genes, from which duplicates are normal and expected. That is why rmdup is not recommended for RNA-seq (or any NGS assay in which duplicate generation is expected irrespective of PCR duplicates).

ADD REPLYlink modified 2.4 years ago • written 2.4 years ago by ATpoint30k

but is there in your opinion a correct solution ? Is the batch effect integration an acceptable way in your opinion ?

ADD REPLYlink written 2.4 years ago by biostar.anon0
1

It's a good solution

ADD REPLYlink written 2.4 years ago by Asaf7.0k
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