Kapa library quantification

[martinjf]

Hello all,

This might be a stupid question but we cannot figure out the answer for sure :
Does the kapa quantification kit quantifies only the P5/P7 ligated fragments or P5/P5 and P7/P7 fragments are also quantified in the same time? I guess only the P5P7 are but I would like to make sure as it is not clear to me reading the documentation.

Thx a lot,

Jef

[pmiguel]

Hello all,

This might be a stupid question but we cannot figure out the answer for sure :
Does the kapa quantification kit quantifies only the P5/P7 ligated fragments or P5/P5 and P7/P7 fragments are also quantified in the same time? I guess only the P5P7 are but I would like to make sure as it is not clear to me reading the documentation.

Thx a lot,

Jef

KAPA just assays the increase in double-stranded molecules during a PCR reaction. Anything that amplifies will be measured by this assay.

It's possible that amplification of P5/P5 and P7/P7 would be inefficient due to "suppression" caused by those molecules forming stem-loops prior to primer annealing/extension. But it is my understanding that suppression PCR reactions need to be fairly finely calibrated.

--
Phillip

[martinjf]

Thank you for your quick answer, I realise I was not specific enough in my question :

we ligate P5 and P7 illumina adapters and as far as I understand it, for protocols not using A-tailing you may have all possible fragment constructions :
1/2 with P5 and P7 adapters
1/4 with P5 adapters both sides of the insert
1/4 with P7 adapters both sieds of the insert

My question is whether the kapa (library quantification kit for illumina) primers will amplify only the construction with the two different adapters or if all constructions will be amplified (I am not concerned about concatemers of adapters). In the last case do you double the quantity of libraries to load your lanes ?

Thx again,

Jef

[pmiguel]

Thank you for your quick answer, I realise I was not specific enough in my question :

we ligate P5 and P7 illumina adapters and as far as I understand it, for protocols not using A-tailing you may have all possible fragment constructions :
1/2 with P5 and P7 adapters
1/4 with P5 adapters both sides of the insert
1/4 with P7 adapters both sieds of the insert

My question is whether the kapa (library quantification kit for illumina) primers will amplify only the construction with the two different adapters or if all constructions will be amplified (I am not concerned about concatemers of adapters). In the last case do you double the quantity of libraries to load your lanes ?

Thx again,

Jef

Normally we don't deal with libraries that have 50% P5/P5 or P7/P7 adapters. Illumina kits use y-adapters that prevent the production of those homo-adapter amplicons.

How were these libraries constructed?

--
Phillip

[pmiguel]

My question is whether the kapa (library quantification kit for illumina) primers will amplify only the construction with the two different adapters or if all constructions will be amplified (I am not concerned about concatemers of adapters). In the last case do you double the quantity of libraries to load your lanes ?

Thx again,

Jef

Yes, the Kapa kit should amplify the P5/P5 and P7/P7 constructs as well as the P5/P7 ones. At least to some extent.

But unless you have done no PCR after your ligation for your library prep protocol, the PCR suppression effect may have drastically diminished the amount of the P5/P5 and P7/P7 constructs relative to the P5/P7 ones. If not, then it is possible the PCR suppression will prevent or limit the kit from detecting the P5/P5 and P7/P7 amplicons.


If you are concerned you could do P5 only and P7 only primed qPCR reactions to determine the concentrations of just the P5/P5 and P7/P7 amplicons.

--
Phillip

[martinjf]

Yes indeed, you answer makes sense.
Thx a lot !

Jef