No amplification with Kapa kit

[rougaroux]

Hello,
I have been using the Kapa Hyper Prep kit to try and prep DNA libraries for target enrichment.
I am using custom Y adapters and dual indexed primers which I know have been used successfully with the Kapa kit before.
My issue is that I am getting virtually no amplification from the PCR. By increasing the number of cycles up to 16, my library concentrations increased by only 2-3X.


The kit I'm working with is new.
I checked my shearing with the bioanalyzer and all samples were within the targeted range (400-600 bp).

I suspect the problem is with adapter ligation...after PCR, I see very high concentrations with a nanodrop (but not Qubit) that went down sharply after bead cleanup. This makes me think lots of adapter dimers and/or unused primers were present.

Has anyone here experienced similar issues with the Kapa kit, or does anyone have any advice on how best to troubleshoot this problem?

[luc]

You could run aliquots of the reaction (before and after bead cleanups) on an agarose gel to see what is going on.
If the ligation does not work porperly, perhaps your samples have some chemical contamination?

[rougaroux]

Thanks for the suggestion, Luc.
Do you know if I would be able to reliably identify unincorporated adapters on a gel?

[luc]

Depends on the dye used and the amount of primers. Ethidiumbrmide is ten times less sensitive for ssDNA compared to dsDNA. The sampe seems to be true for the Bioanalyzer, but such an instrument is more sensitive than our eyes looking at an agarose gel.

[rougaroux]

Okay, well I will give it a try with my post-ligation libraries before and after clean-up and see if there's anything to see.

Does anyone have any other suggestions of common sources of library amplification failure?

[jdk787]

If you're using Nanodrop on PCR products before cleanup, the "high concentration" you saw was likely just unincorporated nucleotides in the PCR mix, so that isn't useful.

If you run your cleaned up post ligation product on the Bioanalyzer you should be able to see a shift to a larger size for your ligated products compared to your original fragmented DNA. Y adapters should make this shift more noticeable since libraries ligated with Y adapter migrate slower through the gel causing them to appear larger than their actual size.

My best guess would be that there's something wrong with your custom adapters/primers or something went wrong with the prep. Maybe add a control sample that uses a non-custom adapter at ligation and p5/p7 primers for PCR.