Adapter Ligation Question

johnmillsbro · 07-01-2010, 10:31 AM

Hello. Looking for some help. We are using solexa to sequence a large pool of PCR products (Vent). Vent doesn't not produce overhangs. Therefore to ligate solexa adapters we will have to either enzymatically add the A overhangs or we would use adapters designed to be blunted directly to our PCR product. My question is: Are the A overhangs essential for efficient adapter ligation? Why do they use the overhangs in the first place?

Thanks many-

John

GW_OK · 07-01-2010, 11:59 AM

Using A tails prevents (somewhat) the formation of adapter dimers and library concatemers. Illumina protocol, though, has a specific A-tailing step after the post shearing end repair so it ought to be a moot point. Are you using some other protocol?

johnmillsbro · 07-01-2010, 12:24 PM

I don't have their full protocol (yet). I just knew you need an A overhang. I am curious why they add the overhang. I suppose it must be to prevent adapter:adapter ligations.

thanks

Bruce E · 07-13-2010, 09:09 AM

The A overhang is also needed so the adapter ligates in the correct orientation. They refer to these as adapters but it is actually one Y adapter so you need it to ligate in the correct orientation and the 3' A does this. If you were using blunt end ligation with blunt adapters as 454 uses you can get many fragments that will have adapters ligated incorrectly.

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07-01-2010, 10:31 AM	#1
johnmillsbro Junior Member Location: Canada Join Date: Jul 2010 Posts: 2	Adapter Ligation Question Hello. Looking for some help. We are using solexa to sequence a large pool of PCR products (Vent). Vent doesn't not produce overhangs. Therefore to ligate solexa adapters we will have to either enzymatically add the A overhangs or we would use adapters designed to be blunted directly to our PCR product. My question is: Are the A overhangs essential for efficient adapter ligation? Why do they use the overhangs in the first place? Thanks many- John

07-01-2010, 11:59 AM	#2
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	Using A tails prevents (somewhat) the formation of adapter dimers and library concatemers. Illumina protocol, though, has a specific A-tailing step after the post shearing end repair so it ought to be a moot point. Are you using some other protocol?

07-01-2010, 12:24 PM	#3
johnmillsbro Junior Member Location: Canada Join Date: Jul 2010 Posts: 2	I don't have their full protocol (yet). I just knew you need an A overhang. I am curious why they add the overhang. I suppose it must be to prevent adapter:adapter ligations. thanks

07-13-2010, 09:09 AM	#4
Bruce E Member Location: Rochester,MN Join Date: Jan 2008 Posts: 15	The A overhang is also needed so the adapter ligates in the correct orientation. They refer to these as adapters but it is actually one Y adapter so you need it to ligate in the correct orientation and the 3' A does this. If you were using blunt end ligation with blunt adapters as 454 uses you can get many fragments that will have adapters ligated incorrectly.