By whole exome or whole genomic sequencing, we can estimate copy number by comparing sequencing depth of a given DNA region vs. that from a pool of normal samples, but for amplicon sequencing, how reliable that approach will be? I heard somebody doing that, but other people argued that the target enrichment either through probe hybridization or PCR, might add confounding factors and make the estimate not so reliable. Does anybody have experience on that?
Your question is very stimulating but it needs some clarification.
If you are doing a vertical comparison inside the same specimens of course this does not make sense. The relative abundance of a single transcript as compared to another one will be biased by PCR efficiency and other variables.
But if you are comparing the same transcript across multiple specimens using the same primers and library preparation strategy the situation is very different. Just as example, if you get in 20 patients the same number of total reads and you observe that in one of the patients there is a huge increase of a determined transcript it is very likely that this gene is amplified in this patient. In this context, whole exome analysis becomes a potent tool to identfy genes (and more importantly gene variants) which are amplified in determined subset of patients. Then, in order to further confirm such results it should be wise to confirm the results with qPCR and related techniques.
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