Two-step PCR in single step?

[JBKri]

We would like to try to do the Illumina two-step PCR (1st PCR with universal tail, 2nd PCR adds indexed adapters) in a single PCR reaction, to lessen chances of contamination. According to the old Roche Amplicon design guide this is a possiblity, although it may create more spurious products. Has anyone tried this? We are wondering how many cycles to use. Normally we would use ~26 cycles in the first round and 8 in the second.

[nucacidhunter]

I would be more concerned with reproducibility which is common with primers having long non-template overhang. To optimise cycle number I would do 30 cycles, run it on BA or similar device and if neccessary do extera cycles to get the required yield.

[JBKri]

I would be more concerned with reproducibility which is common with primers having long non-template overhang. To optimise cycle number I would do 30 cycles, run it on BA or similar device and if neccessary do extera cycles to get the required yield.

What do you mean by reproducibility? The non-template overhang would be the same length if we do the two steps separately, right? I am worried about possible biases, chimeras, long primer-adapter dimers and perhaps other problems that I cannot yet imagine.

Would a good approach be to make sure the inner primer pair will be depleted well before the outer pair?

[nucacidhunter]

I thought you meant to use a long primer pair not two primer pairs. All those interactions are possible but I do not know what the results will be in practice.

[cmbetts]

I've done it, and it works with some drawbacks. It definitely can lead to more primer artifacts do to unintended interactions, and is generally dependent on the 3' end of the tailed primer, so it can need some optimization and not immediately transfer over when adjusting protocols.
The biggest headache for me is needing to qPCR the library because you never know if every fragment has the full adapter sequences or not. My libraries tend to be somewhat nonstandard, and the combination of bioanalyzer and qubit has always been more reliable than the KAPA qPCR kit. The time and inaccuracy of running qPCR combined with more artifacts outweighed any benefits of combining the PCR.

[thermophile]

I'm considering doing this as well but haven't had time to test it out yet. We've had ok results doing the PCR separately but without doing a clean up step between the 2 PCRs. We are getting ~50% of the reads that we expect per sample for the concentration that we are loading which given our level of multiplexing is acceptable.

[JBKri]

....
We've had ok results doing the PCR separately but without doing a clean up step between the 2 PCRs. We are getting ~50% of the reads that we expect per sample for the concentration that we are loading which given our level of multiplexing is acceptable.

We are also considering omitting the cleanup.
Could you explain a little, what do you mean you are getting 50 %? Are the other 50 % dimers, byproducts etc?

[thermophile]

I'm running amplicons, so multiplexing 100+ per miseq run. I just quantify my samples with qubit, so can't tell the difference between primer diamers (and dont' know how well my cleanup is getting rid of them since they're ~180bp)/ partial adapter products and the amplicons with both adapters. But I'm still getting sequences-less than I'd expect if my quantification was perfect, but I only guarantee 10k seqs/sample and I'm mostly hitting that target.

So far I've only been running ~10 samples (~10% of the run) that have been through 2step procedure on a run, the rest of the samples have gone through our regular single step 16s library prep. If I were to start doing lots of 2step maybe I'll look more closely at the necessity of the cleanup between the PCRs. Right now I'm more interested in balancing the workflows between our standard 1 step and rarer 2 step procedures rather than trying to perfect the 2 step on its own.