Qubit versus qPCR

[cmuletz]

Hello,

My question relates to the use of Qubit to qPCR for quantifying your amplicon samples to equimolar ratios -- we have both a Qubit/dsDNA HS Assay Kit and qPCR/ Kapa kit in our lab to quantify 16s rRNA amplicon samples with fusion primers. I will pool ~30 samples together. From looking at the previous threads people recommend qPCR and that is the same with the folks in my lab over Qubit and Qubit over nanodrop. However, most microbiome papers I have found people are using Qubit to quantify before pooling. If most people are doing this then...?

I'm asking for accuracy and time efficiency.

1) How do you feel about Qubit versus qPCR?

2) For qPCR are most people doing absolute qPCR over relative? -- I'm not sure if you need the Kapa Kit to do relative qPCR (you would still need to quantify some samples at some point though).

--relative seems faster with the accuracy -- you have the qPCR results but you don't need to go through the dilutions and replicates to do the absolute qPCR. For absolute qPCR I've been told that you need to do a few dilutions and at least 2 replicates and you need the standards so you can really only quantify 10 samples at a time. I realize with relative you would still need the reps.

--for my 30 samples that is 3 runs on the machine for absolute versus 1 for relative. In the end I will be doing 4 more pools like this.

Any folks with experience your comments would be greatly appreciated.

Thanks!

Carly

[Brunda]

Hello Carly, how did you resolve your problem? I just stumbled onto thread on http://seqanswers.com/forums/showthread.php?t=14511

i wonder if it's possible to do the qubit measurement instead of QPCR (to be sure you have enough sample) and then going for the norm kit.

the advantage of QPCR is that it "counts" only dsDNA fragments with both adaptors ligated, so you can be sure it is specific.

[cmuletz]

I have been using qPCR to quantify molecules/ul of my amplicons. I have not done a test to see how close Qubit readings are to that of qPCR. I was told by lab members that qPCR is much more sensitive. I typically run ~30 samples on one 454 junior plate and have to do at least 2 qPCR runs to quantify all the samples. I do 3 dilutions for the first run (1:2000, 1:4000, 1:8000) to figure out what dilution I really quantify my samples. This of course depends on PCR cycles and starting DNA amount, but I usually use 1:4000 and 1:8000 dilutions to quantify. BUT I do not use the dilutions to do the emulsion PCR. Most of the time I pipette around 1 ul from each sample, and do one large dilution of that sample. Sometimes 2 ul into 20 ml! I have read and heard from others that they just use Qubit readings and it works fine.

[ZWB]

I've been doing 16S amplicon sequencing lately and I've found that quantifying by Qubit and pooling works fine, although not quite as good as qPCR. On most runs I see only a slight (+/- 2%) variation in number of reads from any particular barcode indicating that the multiplexing worked well. Given the extra time and cost of running the qPCR, I don't think it's necessary because it only gives slightly better results. For anything other that amplicon sequencing though I would say that quantification by qPCR is an absolute requirement.