ATACseq_which size for calculation of concentration using qPCR

[EGraf]

Hi everybody,

I have to sequence an ATAc seq pool on our HiSeq. It's the first time I have such a library with broad size range ranging from 150 bp up to 7 kb. I would like to quantify the pool via KAPA lib quant qPCR. I was wondering which average size I should use to calculate molarity in a right way. Average size calculated from the bioanalyzer was 1.5 kb.

For a "normal" library I would use this average size for calculation but I'm not sure in case of the ATAC libs.

Is anyone familial with qPCR and ATAC libs?

Attached please find a bioanalyzer trace of the ATAC pool.

Thanks a lot,
Elisabeth

[nucacidhunter]

Considering that standard KAPA qPCR cycling condition would amplify fragments up to 1kb and also fragments below this size are more efficient in clustering, I would calculate average size between shortest fragment up to 0.9-1kb. Larger fragments will not contribute to quantification or clustering. This works very well when there is only one peak in library. In this case multiple peaks complicate that simple rational. I would cluster at little less (2 pM) than usual to avoid possible overclustering.