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Old 04-23-2017, 10:47 AM   #3
Carcharodon
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Location: Honolulu, HI

Join Date: Jul 2015
Posts: 38
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Quote:
Originally Posted by nucacidhunter View Post
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.
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