RNASeq: Normalization of sample with allele specific duplication

Question

[Ankit]

Hi everyone,

I have general question.

Suppose I have two individuals. One is patient, other is normal. The defect in patient is loss of function of a gene (due to a very small deletion) which results in phenotype.

Surprisingly we also find some Mb duplication at maternal allele in chromosome 1. paternal chromosomes are normal.

In normal individual there is neither a small deletion nor a large duplication. Both paternal and maternal chromosomes are normal.

If we perform RNA-Seq, for these two individuals. Will the Normalization of raw counts follow usual method implemented in any available tools (DESeq2, edgeR or any other) or I have to take care of duplication and follow some other adjustments (which I don't know)?

We would like to perform differential expression analysis between patient and normal individual.

I hope the question is clear.

Please help if someone has a suggestion or prior experience.

Thanks

[Ankit]

Hi everyone, Any help?

[Ram]

Hi,

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Your original post:

[Ankit]

Yes I will take care of this. Thanks

Answer 1

[h.mon]

With one patient and one normal sample, you have a very, very poor design for rnaseq DGE analysis. Other than that, the normalization methods from edgeR and DESeq2 should work for the case you described (except they don't work if you don't have biological replicates). You may have to create a custom reference genome to account for this duplicated region, in case is has some major difference from the reference genome, otherwise counting would underestimate reads mapping to this region.

[Ankit]

Hi Thanks for the reply.

It was just a hypothetical scenario in humans samples.