Quantifying whole genome PCR-free libs without qPCR

[EGraf]

Hi everybody,

I would like to run whole genome sequencing in a high throughput manner.

Libraries are prepared using Illuminas TruSeq PCR-free protocol and chemistry.

The time and cost consuming step is the final quantification with qPCR. I used the KAPA lib quant kit.

I was wondering if the is another method to quantify the final libraries. Just picoGreen seems not to be the best option to get reliable results because we measure also fragments with one or do adapter.

The whole genome libs should be run on a HiSeqX.

Thanks,
Elisabeth

[kmcarr]

The cost of the Kapa qPCR library quant is a tiny, tiny fraction of the full end-to-end cost of your experiment when the HiSeq run is considered. Don't try to save a few dollars by using an unreliable quantitation method to risk thousands of dollars on a failed HiSeqX run.

[kerplunk412]

As you mentioned, any quantification method that only quantifies dsDNA will also include non-ligated or singly-ligated products, so QPCR is the only reliable method for determining concentration of doubly-ligated products in PCR-free libraries. You could theoretically run a "poor man's real-time" using PhiX or the Kapa standards as a reference, but it will be less accurate than QPCR and require more work.

[jlei_face]

Hey EGarf,
My facility is very high throughput so we have moved away from qPCR given both the cost and the time. We do picogreen (Quant-iT) paired with LabChip (similar to BioAnalyzer). Our libraries are sequenced on the HiSeqX also and because there is such a high input concentration compared to other Illumina instruments small concentration discrepancies won't impact the sequencing data.