adapter ligation and library amplification

rjust · 10-31-2016, 12:57 AM

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.

r.rosati · 11-05-2016, 05:46 PM

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.

rjust · 11-11-2016, 01:40 AM

So neither I nor my colleague were completely right. Thanks for the answer and the link!

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
adapter ligation - Illumina	Celia	Sample Prep / Library Generation	5	03-29-2011 11:06 AM
amplification (ligation) bias	tea tree oil	Illumina/Solexa	0	09-08-2010 07:02 AM
Adapter Ligation Question	johnmillsbro	Illumina/Solexa	3	07-13-2010 08:09 AM
Ratio in Adapter-Ligation	mestro2	Sample Prep / Library Generation	0	05-27-2010 03:29 AM
help: time of adapter ligation	lvxiaobao	Sample Prep / Library Generation	1	12-24-2009 06:37 AM

10-31-2016, 12:57 AM	#1
rjust Junior Member Location: Germany Join Date: Oct 2016 Posts: 2	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.

11-05-2016, 05:46 PM	#2
r.rosati Member Location: Brazil Join Date: Aug 2015 Posts: 69	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.

11-11-2016, 01:40 AM	#3
rjust Junior Member Location: Germany Join Date: Oct 2016 Posts: 2	So neither I nor my colleague were completely right. Thanks for the answer and the link!