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Old 03-08-2016, 07:12 AM   #24
DNA_Dan
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Location: Montana

Join Date: Nov 2008
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Assuming the libraries range across the board, Truseq, Nextera, Kapa Biosystems, Nugen, etc., come from a huge variety of DNA/RNA sources, and have different fragment lengths/profiles - How tight can the ddPCR dial them in?

To elaborate, with the Kapa Biosystems qPCR method the tightest we can normalize our pools of samples is 1-3 fold of each other. That is to say sometimes they are pretty even, other times they can vary by up to 3-fold. Can you achieve a better normalization in a pool of very different libraries with completely difference efficiencies using the ddPCR method? If so, how tight? 80% or better? 90% or better?

Cost is a huge pill to swallow because 90K is a lot of rounds of normalization kits and technician time. However I have tried a reiterative process of normalizing, measuring with qPCR, then normalizing again, 2-3 times one after the other and what I have found is that the pipetting error in the dilutions and measurement error have a limit with how close you can "dial" samples with respect to each other. At some point the pools don't get any more "normalized", they actually start to get worse because of the handling error involved or the measurement itself.

So in essence what I am looking for is something that has the accuracy to push the flowcell to it's maximum density reproducibly every time and normalize the pools so evenly you are squeezing every bit of data possible for each sample on every run. We also do a lot of ratio pools (30% one customer, 70% another) that sort of thing. Being able to target this accurately down to 1% would allow us to put more customer samples on a run because we would have the confidence that we would hit our ratio targets more accurately.

Is the QX200 the instrument that can do this? Is this a pipe dream? Where do you feel the QX200 falls short of expectations? What are its limitations?

Ultimately if the method allows for a tighter multiplex/run scenario as I described above, the cost savings of not having to run another run on the sequencer would pay for itself. The main cost and driving factor is still the sequencing reagents.

Last edited by DNA_Dan; 03-08-2016 at 07:14 AM.
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