Normalization In Rna-Seq For Snp Discovery

Question

[ruru]

Hi all.

Is that the good choice to do normalization in RNA-seq to discover high quality SNP? Personally I wouldn't agree with this approach but someone suggest to normalize the samples in order to get high quality SNP. For me we can pool the samples and to discover high quality SNP is from the coverage of sequencing. I need your opinion regarding this.

Thanks.

Answer 1

[Wen.Huang]

are you sure it is not "normalizing RNA", which is to use certain nuclease to degrade highly expressed RNA to make coverage more homogeneous.

Answer 2

[ruru]

according to them, normalization is referring to expression stages for each samples. so, does normalizing RNA contribute to highly expressed RNA?

[Arun]

I still am confused as to what you refer to as normalization. What do you mean by "expression stages"? If it is during library preparation, then its what Huang already mentioned. If its regarding gene expression, then normalization tries to correct for biases in gene expression, expressed as a measure of read counts, that are introduced due to inherent GC bias, amplification bias, poly-dT/random priming, gene length etc..

For SNP detection, normally people remove duplicate reads and then call for SNPs. I guess this is what you are calling as normalization? If so, yes, I believe that SNP calling should be done on reads after removing duplicates.