Why sub-saturation qPCR in ATAC-seq but not other seq methods?

[kropak1807]

Hi everyone,

I have been asking around and nobody seems to have a sufficient answer for my question. In ATAC-seq we run a side qPCR reaction in order to determine the appropriate number of PCR cycles to amplify the library without reaching saturation. Why is this only used in ATAC-seq and not other sequencing techniques? Seems to me like it should be used in all sequencing techniques, especially those that are sensitive to PCR duplicates.

Thanks

[nucacidhunter]

I think in ATAC-seq quantity of available DNA in different experiments can be highly variable requiring experiment specific optimisation, while in other libraries such as RNA-Seq or DNA libraries input is normally fixed or is in a small range and pre-optimised PCR conditions can be used.

[NickPantelireis]

I can't seem to figure out if when doing ATAC-seq you need to maintain the selected cycles the same throughout samples? For example I have 4 samples and I'm doing n=3. So do I select the number of cycles for each of the 12 samples plus replicates, do I select cycles for each sample and keep constant in replicates or do I run one qPCR and select the lowest No of cycles for all samples?