[austinso]

Really? That's interesting news indeed. I had figured that with all the extra chemicals to keep the droplets from merging and the necessity to keep the droplets at exactly 1nL, the costs would be significantly higher than what we already have. I suppose though that ruling out the very costly standard series is a major advantage to ddPCR.

I agree on the additional advantages of ddPCR. It's very useful for some applications.

They didn't do a good explaining, did they? The cost is about U$5 per well. For library quant you really need 4 dilutions to measure, where the last two are likely to be within the dynamic range of the instrument (<100,000 copies/ul or <0.16 pM).

Could you possibly provide us with some data indicating this? The data that Bio-Rad showed was good, but not stunning in my opinion. They showed us a graph with Cluster Density PF for samples quantitated by both qPCR and ddPCR. Sure, the error bar for ddPCR was significantly smaller, but it was still very much present. What would you define 'extremely well'?

I'd like to but I don't know if I can. Basically, you can plot cluster density against input concentration determined by ddPCR, and get a nice curve. Between 5 pM and 10 pM you will essentially get the same number of Q30 reads on a Miseq, though the %PF may decrease. That may have changed with the V2 chemistry though. I'm assuming the same is true for the HiSeq.

The error is due to dilution error, and extrapolating back to the starting concentration. You can get a sense of this by pipetting water onto an analytical balance, and measuring the average mass dispensed to give a sense of your pipetting accuracy and more importantly, bias. This doesn't account for wetting properties of the tip plastic for the DNA solution.

Would you mind pointing me to such a program? I would be very much interested in comparing it with my own method. Thanks in advance for any help provided.

Sure. In fact the one I use(d) the most was LinRegPCR, which is from a group in the Netherlands.

A good resource for qPCR (though hard to navigate) that will link to various methods of analysis is here

Best

Austin