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-   -   ddRAD: Performing PCR BEFORE Size-Selection? (http://seqanswers.com/forums/showthread.php?t=75632)

Carcharodon 04-22-2017 02:49 PM

ddRAD: Performing PCR BEFORE Size-Selection?
 
Greetings!

A quick search yielded a thread asking a similar question, but it went unanswered.

Up-front, my question is: why perform a fragment size-selection before performing PCR, and has anybody here done it the other way around (PCR before size-selection for ddRAD or a similar protocol)?

Intuitively, I understand that having fragments of the proper/desired size means potentially greater amplification efficiency at that particular size (if only 200 - 300 bp fragments are available, PCR will act only on these and not fragments of other sizes because they don't exist).

However, I'm going to perform a PippinPrep size selection, and the Y-adapters will force me to select a "larger" size fragment than what I really want, because the Y-shaped adapter will slow migration through a gel. I'm looking to collect ~ 250 - 300 bp fragments (w/o adapter), and I have yet to see a good, reliable way to select fragments of this size (do I tell the machine to select 400 bp fragments? 500 bp?).

An obvious way to get around this problem is to perform PCR first, which will rid my fragments of the Y-shaped adapter, making size-selection a lot more straightforward. And I have seen folks on this forum recommend doing just that.

So...has anyone here tried this? What are some considerations I should keep in mind if I were going to try this approach?

Many thanks,
Sean

nucacidhunter 04-22-2017 06:24 PM

Only P2 adapter in ddRAD has short non-complementary overhang so its effect on anomalous migration of ligated fragments in microfluidics separation devices such as Bioanalyser is less pronounced in comparison with standard forked adapters. This effect also is minor on agarose gels and causes a small shift in size-selection on Pippin devices. You need to specify size selection range considering added adapter length. If your library needs precise size range you may need to do a trial to adjust for the size shift.


Advantages of size-selection in ddRAD before PCR:
1- Low mass of DNA overcoming using multiple lanes for size-selection
2- Less bias in PCR amplification and therefore more uniform tag coverage because of uniform fragment sizes

Carcharodon 04-23-2017 11:47 AM

Quote:

Originally Posted by nucacidhunter (Post 206613)
Only P2 adapter in ddRAD has short non-complementary overhang so its effect on anomalous migration of ligated fragments in microfluidics separation devices such as Bioanalyser is less pronounced in comparison with standard forked adapters. This effect also is minor on agarose gels and causes a small shift in size-selection on Pippin devices. You need to specify size selection range considering added adapter length. If your library needs precise size range you may need to do a trial to adjust for the size shift.


Advantages of size-selection in ddRAD before PCR:
1- Low mass of DNA overcoming using multiple lanes for size-selection
2- Less bias in PCR amplification and therefore more uniform tag coverage because of uniform fragment sizes

Thanks for your reply. I think, since I'm going to use single-end Illumina reads, that it would be better to select slightly larger fragments overall. So I may try to overshoot just a bit. Of course this means that my number of loci may be affected, but since this is all (educated) guess-work so far, I suppose it's no big deal.

I know that PCR will be biased toward smaller fragment sizes, and that's not too much of a problem, because I'm targeting fragments at the lower end of my range. And I think that bias could be overcome in part by increasing extension times.

I'm intrigued by your first point; I know Pippin-Prep has a volume limit, but is there a concentration/mass limit for DNA? It never occurred to me that having too much DNA was possible. Perhaps I need to read the documentation more closely!

I think I'm relatively convinced that I should perform size-selection before PCR. When I did this before, a couple of my libraries did amplify beautifully, so I know it works. The failures have made me a bit gun-shy. :D

nucacidhunter 04-23-2017 05:12 PM

Quote:

Originally Posted by Carcharodon (Post 206625)
When I did this before, a couple of my libraries did amplify beautifully, so I know it works. The failures have made me a bit gun-shy. :D

I wonder by "failure" you mean that a digested-ligated DNA PCR was successful without sized-selection and failed after size-selection. If that is the case I would column purify Pippin eluate or dilute it for PCR or use different DNA polymerase because Pippin buffer composition can inhibit some polymerases activity.

Carcharodon 04-23-2017 09:19 PM

Quote:

Originally Posted by nucacidhunter (Post 206626)
I wonder by "failure" you mean that a digested-ligated DNA PCR was successful without sized-selection and failed after size-selection. If that is the case I would column purify Pippin eluate or dilute it for PCR or use different DNA polymerase because Pippin buffer composition can inhibit some polymerases activity.

Well, I originally followed the protocol laid out by Peterson et al. (2012), where digested DNA with ligated adapters were size-selected (w/ Pippin-Prep) and then went through PCR.

3 or 4 out of my 6 total libraries showed amplification. The other two did not. So the eluate itself didn't seem to be inhibiting PCR. I suspect that there was just very little DNA in the size-selected DNA for those 2 - 3 libraries. Which is one reason I was considering running a PCR before size-selection.

I'm taking steps to ensure that won't be the case here, but you never know. At least now I have the benefit of experience!

nucacidhunter 04-23-2017 09:42 PM

You can use 5% (or any other quantity depending on your workflow) of digested-ligated DNA to do a PCR and resolve amplicons. It should show the size region with poor amplification so you can avoid those regions during size-selection.

ymilesz 05-01-2017 11:57 AM

Quote:

Originally Posted by Carcharodon (Post 206629)
Well, I originally followed the protocol laid out by Peterson et al. (2012), where digested DNA with ligated adapters were size-selected (w/ Pippin-Prep) and then went through PCR.

3 or 4 out of my 6 total libraries showed amplification. The other two did not. So the eluate itself didn't seem to be inhibiting PCR. I suspect that there was just very little DNA in the size-selected DNA for those 2 - 3 libraries. Which is one reason I was considering running a PCR before size-selection.

I'm taking steps to ensure that won't be the case here, but you never know. At least now I have the benefit of experience!

Hi I am also following the ddRAD protocol from Peterson et al 2012, and I too am having trouble with PCR after size selection. Are you doing the 20uL reactions as suggested as well?

Carcharodon 05-15-2017 04:30 PM

Quote:

Originally Posted by ymilesz (Post 206887)
Hi I am also following the ddRAD protocol from Peterson et al 2012, and I too am having trouble with PCR after size selection. Are you doing the 20uL reactions as suggested as well?

Hi ymilesz,

Sorry for the late response. At first I actually ran 50uL reactions (due to low DNA concentrations in the elute), and then I tried running 25uL reactions.

Interestingly, the ones that tended to fail were the ones that showed higher DNA concentrations post-size-selection. I added water to these to dilute them down to 20ng total DNA content. I'm starting to suspect that this may have affected my results, but I'm not sure.

nucacidhunter 05-16-2017 12:48 AM

I think using 250 pg of size-selected DNA per ul of PCR will be enough. If only 0.25% of the tags are amplifiable, after 12 cycles yields will be over 2 ng/ul.


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