heat denature or not?

[ilma]

Hei,

I have fusion amplicons for one way sequencing (designed to be used with emPCR lib-L kit). Does one need to heat denature DNA before adding to Capture beads as it is described in emPCR lib-L protocol?

[behavin]

I would say it would be a safe bet to heat denature, as you'll likely get lower than anticipated enrichment otherwise.

[ajthomas]

You don't need to denature ampicons of that sort. You only need to denature the library if both strands can function in emPCR, which is the case with libraries prepared by ligating on rapid library adapters. Those adapters are Y-shaped, with the A adapter on one strand and the B adapter on the other strand. Therefore, both strands have an A and B adapter and both can serve as a template for emPCR. Amplicon libraries made with fusion primers have the A and B sequence on one each, so only one strand can function in emPCR.

You can denature if you want, but it won't make any difference.

[pmiguel]

You don't need to denature ampicons of that sort. You only need to denature the library if both strands can function in emPCR, which is the case with libraries prepared by ligating on rapid library adapters. Those adapters are Y-shaped, with the A adapter on one strand and the B adapter on the other strand. Therefore, both strands have an A and B adapter and both can serve as a template for emPCR. Amplicon libraries made with fusion primers have the A and B sequence on one each, so only one strand can function in emPCR.

You can denature if you want, but it won't make any difference.

I think you are missing a subtlety here. The salient issue is whether both strands of the ds template are full "strand" and "strand complement". In essence, are both strands going to generate the same template to be captured by the bead, or will they each generate a different template.

Y adapters create 2 different library molecules -- one in one orientation (with respect to the A adapter), the other in the other orientation. They are annealed together in the center, but upon emPCR will generate two different templates. When sequenced these should produce discordant sequences -- hence should produce a mixed bead 100% of the time.

The non-Y adapter templates do not have this issue. So you do not need to denature them. However if you do denature them, your CPB should effectively double. Right? Two strands, either can be amplified in an emulsion reactor.

We just denature everything now. Because if you have amplicons you will probably have some primer dimer annealed to your full length amplicons. I would rather peel this off into its own reactor where it will ruin that bead, but will allow the full length amplicon to produce a good sequence.

Of course the denaturation can be problematic. If you do it in a small volume, you can end up throwing you concentration way off because of evaporation.

--
Phillip

[ajthomas]

I think you are missing a subtlety here. The salient issue is whether both strands of the ds template are full "strand" and "strand complement". In essence, are both strands going to generate the same template to be captured by the bead, or will they each generate a different template.

Y adapters create 2 different library molecules -- one in one orientation (with respect to the A adapter), the other in the other orientation. They are annealed together in the center, but upon emPCR will generate two different templates. When sequenced these should produce discordant sequences -- hence should produce a mixed bead 100% of the time.

The non-Y adapter templates do not have this issue. So you do not need to denature them. However if you do denature them, your CPB should effectively double. Right? Two strands, either can be amplified in an emulsion reactor.

Well, yes, that's basically what I was saying; you just stated it more explicitly. In the case of the amplicon, only one strand will function in emPCR because only one strand has the B adapter while the other has the reverse compliment of B. That second strand will, however, produce a copy that can then bind to the bead in the second round. Molecules with the Y-adapter will produce two strands (of different sequence) that can bind the bead in the first round.

And yes, the Y-adapter will effectively double the cpb, although such a comparison requires 100% efficiency in the library prep. In practice, amplicon libraries produced with fusion primers will be used at a lower cpb ratio and will be more consistent because there is no ligation step, and every molecule will be functional (assuming the primers are good quality and all full length).

We just denature everything now. Because if you have amplicons you will probably have some primer dimer annealed to your full length amplicons. I would rather peel this off into its own reactor where it will ruin that bead, but will allow the full length amplicon to produce a good sequence.

Of course the denaturation can be problematic. If you do it in a small volume, you can end up throwing you concentration way off because of evaporation.

--
Phillip

I hadn't really considered the idea of primer dimers annealed to the molecules. In my own sequencing, I haven't any evidence that that happens frequently, but I suppose it's possible. It would be interesting to an experiment to see if one really does get fewer mixed/dot reads if the amplicons are denatured first.

I've also thought about the evaporation issue, and for that reason I have generally avoided denaturing amplicons. I've always figured that if it isn't needed, don't do it because that's just one more opportunity to introduce variability or for something to go wrong.