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Hi Everyone, my name is Efi Melista and I’m the Head of Field and Lab Operations at Omixon.
And I tend to go to a lot of labs to train people on NGS for HLA typing, using Holotype HLA,
and very often one of the questions that a lot of people ask is what is the difference
between qPCR versus Qubit for library quantitation?
So I figured we should create a little video to discuss all of this.
So today we will be talking about should we do qPCR for library quantitation, or should
we do Qubit for library quantitation?
What is better?
What is the difference?
What should you use?
The straight answer is that they’re both the same.
Well… they work differently, but essentially they give you the same kind of information.
They both measure the concentration of your library and help you guide how much you dilute
to load on the MiSeq.
The important thing to remember is that you always want to make sure that the concentration
that you’re measuring of your library is accurate enough in order to do an accurate
dilution because if you overload the MiSeq, it will end up in over-clustering; if you
underload the MiSeq, it will end up in low cluster density, which means that you will
not generate enough data for all of the samples and loci that you are running.
So it is very important to have an accurate measurement.
So what’s the difference?
The qPCR is able to quantify the library using Illumina-specific primers.
The kit rather, that we use, the Kapa Biosystems kit, it uses Illumina-specific primers that
are complimentary to the Illumina-specific adaptors, that are already ligated on the
indexed libraries.
So this way it is able to measure how much is the adaptor-ligated library – how much
is the adaptor-ligated material that you have in your sample.
So it’s a very-very accurate measurement, so when you dilute that down, to a certain
concentration, you know that you’re diluting a very accurate measurement.
So what’s the difference with the Qubit then?
The Qubit on the other hand, it uses a double-stranded DNA fluorescent dye, which is pretty much
an equivalent to a SYBRgreen dye.
What this means is that it intercalates to anything double-stranded in your sample.
So it is able to measure anything that is double-stranded in your library.
Is this an accurate measurement?
It’s not as accurate and specific, as the qPCR.
But is that an incorrect measurement?
Actually it’s not.
And the reason why is because when you enter that value into the excel workbook that we
provide with the Holotype HLA kit, then you can actually calculate very accurately what
is the true amount of your library in your sample.
In the workbook, for this calculation, we actually use a standard curve in the background
that has been created with more than a hundred different libraries that have been quantified
both with qPCR and Qubit.
So that helps remove any variability in this calculation.
So it’s very convenient that way.
So you’re going to tell me “which one should I use”?
Well, you can use either one: the Qubit will help you save time, the qPCR, however, is
a bit more specific.
The qPCR, I would say it is more informative when you’re in a troubleshooting mode.
So if you have figured out that something went wrong into the process, into your whole
workflow, the qPCR can actually help you give you more information to tell you where did
things go wrong in a way.
So it’s a bit more informative.
But for good, high-quality libraries, either one will do.
So it’s up to you, the choice is yours!
If you want to know more about it, feel free to contact us at support@omixon.com or make
sure you visit our website, omixon.com and the MyHolotype registration area, where you
can actually find a lot more information.
Thank you for watching!
Bye!