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Old 08-01-2017, 01:21 PM   #110
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by Brian Bushnell View Post
We have several theories for what was driving this on HiSeq... the most plausible being something like, "library A had too many unincorporated adapters,
Yes, is likely to be an issue.
Quote:
Originally Posted by Brian Bushnell View Post
library B had too many adapter-free inserts, and after mixing them, library B adopted some of the free adapters from library A".
No, adapter-free inserts will not be joined with unicorporated adapters without the intervention of a ligase.

Remember, DNA can be converted back and forth from single-stranded to double-stranded without the intervention of any enzyme if the right temperature/salt/concentration is present. The hydrogen-bond-guided interactions between the bases of reverse-complementary strands of DNA are reversible under these conditions.

The process of breaking the phophodiester/ribose backbone requires much more energy. Joining DNA strands via their backbone pretty much requires an enzyme.
Quote:
Originally Posted by Brian Bushnell View Post
Which would indicate that it involves both the donor and recipient library. But I'm not sure if that mechanism is important for NovaSeq.
Probably the same. Seems like the only major difference is that you don't have to add the Ex-Amp glop to your denatured sample when using the NovaSeq. That happens in the instrument.

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Phillip
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