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Old 10-31-2016, 12:57 AM   #1
rjust
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Default adapter ligation and library amplification

Hello all! I was discussing Ion Torrent library preparation (Ion Plus Fragment Library Kit) with a colleague and noticed that I might have previously misunderstood some things about the process. After reading up a little, am I understanding this correctly?

During library preparation both A and P1 adapters are ligated to the amplicons. Since this is random the ratio of correctly formed A--fragment--P1 combinations and incorrect ones (A--fragment--A or P1--fragment--P1) is basically 50/50. This 50% ratio of undesired products stays the same through library amplification because all combinations are amplified equally. The difference is only in the emulsion PCR step because the incorrect adapter combinations won't be amplified here and will (hopefully) fall away during the template enrichment process.

Previously I'd thought the library amplification step would reduce the ratio of undesired combinations, but that would only be true when using tailed adapters like with Illumina or some other strand-specific selection technique.
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Old 11-05-2016, 05:46 PM   #2
r.rosati
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Not quite: supposedly, the combinations will not be amplified equally. Fragments that ligated the same A or P1 ds-adapter on both ends will have reverse-complementary ends. So after denaturation, while temperature is lowering to Tann, these single-stranded, adaptor-ligated DNAs are prone to form a hairpin that can impair amplification. So "A-f-A" o "P1-f-P1" molecules should amplify with a lower efficiency than "A-f-P1" fragments.

Last edited by r.rosati; 11-05-2016 at 05:55 PM.
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Old 11-11-2016, 01:40 AM   #3
rjust
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So neither I nor my colleague were completely right. Thanks for the answer and the link!
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