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  • #16
    We're just about to test BioRads ddPCR method. Will let you know how it goes!

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    • #17
      The ddPCR method is pretty good...but just thought I'd mention a clever twist:
      Attached Files

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      • #18
        I know this thread is a bit older now, but I am interested in other alternatives as well. Kapa is darn expensive, but IS consistent. I think I'll need to play with different concentrations of primer, along with different kits, including the kapa universal Fast Sybr to find an alternative Sybr green kit to reduce cost and keep consistency.

        One thing I am wondering: Does anyone have a clue as to how the Kapa standards are not degrading at such low concentrations (lowest is 0.0002pM)? If i remember correctly, DNA under 1nM is susceptible to degradation even in -20 storage. My concern is that, even if I amplify my own library, kapa standard, or PhiX standard, that it will degrade if I keep stocks of the lower concentrations. I can do a serial dilution, but I'd rather be able to keep premade stocks in a PCR strip so that I can use a mutlichannel for qPCR setup. Do you think there are any modifications? I tried to amplify a Lifetech taqman standard one time and was unable to get a clean product, and I wonder if it was due to the design of it, rendering it incapable of being amplified.

        Would it make sense to add a phosphorothioate bond into my primers if I attempt to amplify either the kapa or PhiX in order to reduce degradation? Do those bonds protect only oligos, and not an entire amplicon?

        Has anyone else come up with a reliable method for quantification of a pooled library other than using kapa kits?

        Thanks for your opinions!!
        Jeremy

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        • #19
          Originally posted by JeremyDay View Post
          One thing I am wondering: Does anyone have a clue as to how the Kapa standards are not degrading at such low concentrations (lowest is 0.0002pM)? If i remember correctly, DNA under 1nM is susceptible to degradation even in -20 storage.
          "susceptible to degradation"? What on earth do you mean?

          Not sure who told you this, but it sounds like some sort of folklore invented to explain why yields, etc. are so poor (percentage-wise) when the amount of sample is very low.

          I have a better folktale for you: the reason this happens is that everything has a binding capacity for macromolecules. For plastics this binding capacity is probably mostly fairly low -- such that at ug levels, it is not discernible. But the smaller amount of a macromolecule you process, the more noticeable loss to this sort of binding will be.

          Mitigating against these sorts of losses would involve using low bind plastic and/or spiking your samples with a detergent.

          --
          Phillip

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          • #20
            Anyone have any feedback on the Biorad QX200? Does it work as advertised for NGS quantitation or is this just "smoke and mirrors"? Seems like the next logical step to take.

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            • #21
              Old thread but same issues with qPCR. I am surprised that no one is jumping on the ddPCR bandwagon. I guess it just has it's own issues and isn't that popular. Anyone with experience care to comment? Desperately looking to ditch qPCR.

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              • #22
                Originally posted by DNA_Dan View Post
                Old thread but same issues with qPCR. I am surprised that no one is jumping on the ddPCR bandwagon. I guess it just has it's own issues and isn't that popular. Anyone with experience care to comment? Desperately looking to ditch qPCR.
                Yep, qPCR can be a real headache. I guess the major issue with ddPCR is the instrumentation cost - $90,000 for a decent system. Once that price comes down, then it'll see more adoption. Just my 2 cents.

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                • #23
                  Originally posted by cement_head View Post
                  Yep, qPCR can be a real headache. I guess the major issue with ddPCR is the instrumentation cost - $90,000 for a decent system. Once that price comes down, then it'll see more adoption. Just my 2 cents.
                  Yes. That is a huge barrier. That being said, cost per sample will be less than KAPA, since you only need to dilute and measure the sample at 1e-6 to get number of molecules. No standards required. You would load 3.3 billion molecules to get 1000K/mm2.

                  The caveat is that if you have too much unligated adaptor and/or primer carrying over through your purifications it convolutes the measurement and leads to an overestimate. Still you can more readily quant and correct than qPCR.

                  If your libraries are fairly clean, Qubit is actually good enough.

                  The real advantage is when you need to multiplex across samples per run.

                  Best

                  Austin

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                  • #24
                    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, 08:14 AM.

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                    • #25
                      Just curious, what instruments are you all using for the KAPA qPCR? We are using our QIAGEN Rotor-Gene Q and have enjoyed some pretty good success. We also have the option of using a 7500 Fast Dx and I was wondering if anyone else has used this?

                      Thanks!

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                      • #26
                        Originally posted by buckiseq View Post
                        Just curious, what instruments are you all using for the KAPA qPCR? We are using our QIAGEN Rotor-Gene Q and have enjoyed some pretty good success. We also have the option of using a 7500 Fast Dx and I was wondering if anyone else has used this?

                        Thanks!
                        CFX Connect (Bio-Rad) works well. We also have a Corbett RotorGene 3000 (much older version of the "Q"), and it works on that as well.

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                        • #27
                          We're using an older Applied Biosystems 7900HT.

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                          • #28
                            Originally posted by DNA_Dan View Post
                            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?
                            Within chemistries, pretty good. We can certainly dial in 1000K/mm2 when the libraries are produced with a standardized workflow.

                            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?
                            I think that within libraries from a given chemistry, you should be able to be within 20% of each other. The biggest reason this is the case is that you are measuring number of molecules directly, not inferring from a standard curve with a size correction. The other advantage is that you can get a better handle of concentration in the presence of primer/adaptor carryover, which you just cannot from qPCR. You can also see whether you likely have empty libraries or one with nice inserts.

                            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.
                            Absolutely. The 90K price is steep. Particularly if it was only used for library quant. But that's really not the point of the instrument.

                            And absolutely. Any measurement is only as good as the technique used to create the sample to be analyzed. ddPCR is not a panacea of accuracy. I played with a bunch of methods for creating those dilution series, but at the end of the day well-calibrated pipettors and consistent operator pipetting of 1:100 dilutions were the key. And if there is a consistent bias in technique, that can be accommodated in the calculations.

                            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.
                            Well...it is a pipe-dream to believe that any analytical measurement with manual handling steps can have 1% variance. Error is compounded over the number of handling steps.

                            If you are a high throughput lab, and you can use a full plate more often than not, the cost per sample is very reasonable. The minimally optimal format cost-wise is in groups of 8. If you do a lot of one offs, the cost per sample becomes higher, mainly because of the peripheral consumable costs. On that scale the QuantStudio may be better.

                            From a workflow perspective, once you get the hang of it, it is trivial. And if all you have to do is get the right normalization is do a single dilution series and a single measurement (or two...I usually do 1e-6 and 1e-4), then that also has value.

                            Best

                            Austin

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                            • #29
                              NEBNext versus Kappa library quant kits?

                              Hi

                              this is my first post to seqanswers and I thought it belonged in this thread as it's related. I was using Kappa kit to quantify PCR-free library preps but switched to NEBNext library quant kit as it is very much cheaper. However, it seems very variable to me (compared to Kappa) and I was wondering if others had tried this kit and what your experience of it was? Thanks

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