Is end repair necessary if I use Taq pol and PNK?

[Chr15]

Hi,

We are hoping to carry out NGS of PCR amplified DNA aptamers (80 bp). We have PCR’d up the DNA aptamers using Taq pol (which should generate a 3’ A overhang) and then treated using PNK (to generate a 5’ phosphate). As I understand it the DNA is now in the correct configuration for adaptor ligation (using the Tru Seq PCR free kit), is this correct? The alternative is to end-repair the PCR products and A-tail, but is this necessary if I use the Taq pol – PNK method as above?

Any advice much appreciated

C

[pmiguel]

Should work.
You could buy phosphorylated primers and skip the PNK as well.
--
Phillip

[kerplunk412]

I tried something very similar to this recently, and it worked much worse than I expected. It looked like I had a lot of blunt-ended inserts that made crazy concatemers, which leads me to believe the PNK treatment worked well but the Taq did not add A overhangs as I thought it would. I went ahead and did the end repair and A-tailing and then the ligation worked as expected. I was surprised, but the data was pretty clear.

[Chr15]

Thank you for taking the time to respond, much appreciated!

Using the Taq pol – PNK method to produce our DNA product ready for adaptor ligation, we get only a tiny amount of ligated adaptor product, so it’s reassuring to hear that this method has been problematic for Kerplunk412.

I think it’s time to move onto the end repair and A-tailing method……..

….. which brings me to another question.

Is it ok to continue using TaqPol to PCR up my DNA ready for adaptor ligation if I switch to the end repair and A-tailing method or should I be using an enzyme which doesn’t leave a 3’ overhang (Pfu) ? The PCR is optimised with TaqPol, but I am concerned that as this already leaves a 3’ A overhang, will the end repair process remove (which I want) this or add a corresponding T on the other strand (which I don’t want).

Much much appreciated folks!

C

[cliffbeall]

Couldn't you add the Illumina adapter bits by overlapping PCR, as done in 16S amplicon and Nextera protocols?

[kerplunk412]

My results indicated that most of my PCR products were blunt ended, but it is possible (although unlikely) that occurred during the PNK treatment. There should be no problem with having an extra A on the ends of the reads for alignment and data analysis purposes, and as long as <50% of the products have the A I also don't think it will be a problem for sequencing. That being said, it may be worth it to optimize your PCR with another polymerase, as this should really only involve a gradient PCR or two. Also, lower error rates are never a bad thing.

Overlapping PCR is also a good option, but may require more optimization than you would think. Overlapping PCR is ideal for high sample numbers, because when processing hundreds of samples it is much less expensive per sample than ligation-based library prep. So if you have a lot of samples it is something you may want to think about.