[pmiguel]

What did your library look like when you did the quant before you loaded it?
We find that if there are lots of small fragments they will preferentially cluster and produce poor quality results. I've also seen a similar issue when there are fragments significantly longer than those we are looking at. Basically anything outside of the 400bp-800bp range is an issue.

So, I don't know if I should even bring this up. It seems to be pretty far out of most lab's comfort zone.

But native DNA electrophoresis hides a critical issue we see with some substantial fraction of amplicon pools submitted to us. We see cases where strand-denatured assays (eg, heat denature the sample and run it on an RNA pico chip) show lots of short fragments that barely show up on a non-denaturing (DNA High Sensitivity). So I presume the short fragments are annealed to full-length fragments.

We mainly decided to start using pico chips to check libraries for NovaSeq runs -- index hopping being potentiated by primers/primer-dimers -- or at least that is what we are told. But then we saw that this assay could predict bad amplicon pool runs we started using it for that purpose as well.

I don't see why you couldn't develop the same denatured DNA run on an RNA assay for the multiNA. We just heat the DNA to 96oC for 2 minutes in a thermal cycler with a heated lid to prevent evaporation. Then we "snap cool" the DNA in a wet ice bath before loading.

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Phillip

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Phillip