Extra-peaks in melt curve in KAPA quantification

[AnnaMC]

Hello,

In the Q-PCR results from KAPA quantification of my libraries, I got extra-peaks in the melt curve graph and I can not figure out the reason. I have attached the image

- I used the TruSeq stranded total RNA sample preparation with the Ribo-Zero depletion and the BA didn´t show any anomaly (according to tehnical support the extra peaks could be adapter-dimer but that is not consistent with the BA profiles).

- The samples are primary cells from paraganglioma and glioblastoma tissue and, theoretically, they have presence of cytomegalovirus (that is what we want to confirm).

Does anyone have a similar experience and an explanation, please?

Thank you in advance for any help!

Anna.

[bunce]

Hi AnnaMC,
Reading melt curves without all the information is a bit like reading tea leaves but I will have a shot ;-)

Is there a no template control in here? I ask as this is the most obvious way to ID dimer as opposed to bona fide product. Could also run a gel to see.

Is this run inconsistent with your previous kappa runs?

Are the CT values tracking correctly for a dilution of your library?

If these products are amplicon, and not dimer (the melt temps might suggest this) then the most obvious explanation might be that you have length and/or sequence differences in the library you are preparing. Even a few bases can bump the mean melt temperatures around a few degrees.

Dimer for P5/P7 (which is what you are quanting with in the kappa kits) would typically be a small-ish product. It may be that you have a short cDNA artefact from your library build?

Hope this helps - more questions than answers I am afraid but I had a bash.
Cheers.

[nucacidhunter]

I have not done any qPCR melt curve for library quantification. I do not think in this application it is required or would give any information as most libraries are pool of heterogeneous fragments with each having its own Tm unlike expression analysis or SNP typing where amplicons are homogenous or have very similar sequences. If you don’t see any primer-dimer peak in BA traces then it should be fine to proceed with sequencing.

Edit: Low melt peaks could be from primer-dimers formed during qPCR from residual adapters or primers (or combination) used for qPCR not from the library. Running qPCR amplicons on BA should show some primer-dimers if this is true.

[jdk787]

I don't think melt curve analysis is all that valuable for determining library quality.

I've seen somewhere that Kapa says that an extra higher peak is an indication of adapter dimer, but if you don't see any dimer on your BA trace I wouldn't worry too much about it.

[pmiguel]

I would emphasize what most have already said: "What on earth makes you think that you should have a tight size distribution in the melt curve of a library?" That melt curve is there for single PCR product testing for qPCR. For a library it doesn't really indicate anything because you expect your products to be a mixture of different GC%.

--
Phillip

[AnnaMC]

Hello everyone,

Thank you very much for the inputs and a huge apology for my late reply!

bunce, I guess it was not easy to try to find an explanation when there was not enough information from my side but thanks!

nucacidhunter, the samples were run anyway but it was interesting to try to know about this profile.

pmiguel, I am working with a group with long expertise in Seq and, when I run KAPA quantification in my samples, it was the first time for them to see these extra peaks. My work is focused on the study of a virus with an unique behavior therefore, from my point of view, it was necessary to look into each step more carefully.

Anna.