SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Very low efficiency adapter ligation with amplicons and PCR free Kurt Lamour Illumina/Solexa 15 10-02-2015 08:18 AM
efficiency of end repair, A-tailing and adaptor ligation ychang Sample Prep / Library Generation 0 05-19-2014 01:48 PM
Adapter trimming NEBNext Library / MiSeq foolishbrat Bioinformatics 8 01-27-2014 01:06 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

Reply
 
Thread Tools
Old 06-12-2015, 01:09 AM   #1
mbk0asis
Member
 
Location: Korea

Join Date: Dec 2011
Posts: 41
Default NEBNext Adapter Ligation Efficiency

Hello.

Has anyone tried "NEBNext Multiplex Oligos for Illumina" to generate sequencing libraries? I have been experiencing a very poor ligation efficiency of NEBNext (hairpin) adapter. I ligated end-repaired/3'A added 1 ug of sonicated gDNA to NEBNext adapters or TruSeq adapters and amplified them by 18 cycles of PCR. The result was frustrating. The NEB one was barely amplified, whether "USER" was added or not, compared to TruSeq samples. Is there something I should've consider for the hairpin adapter ligation? If any of you has experienced this, help me please!!!


<NEBNext Oligo kit I used>

mbk0asis is offline   Reply With Quote
Old 07-11-2015, 12:06 PM   #2
IdahoRAD
Junior Member
 
Location: Idaho

Join Date: Jan 2014
Posts: 9
Default

When I use these kits, which I really like, I just use the TruSeq adapters. Unless you specifically NEED the NEBNext adapters, why not just use the TruSeq?
IdahoRAD is offline   Reply With Quote
Old 07-12-2015, 02:44 AM   #3
nanos
Member
 
Location: Vienna

Join Date: May 2010
Posts: 11
Default

Hi,

we always use the NEB adaptor and in our hands it is clearly superior in terms of ligation efficiency compared to the Standard Y-shaped adaptors.

I don't know the current version of the protocol. Back in the days, the USER digest was done in the first step of the PCR.
We changed that in a way that we do the USER digest for 30' to 1h at 37C, purify the DNA with Ampure beads and then perform the PCR.

That clearly improved the yield of our libraries.
nanos is offline   Reply With Quote
Old 07-17-2015, 09:53 AM   #4
ScienceGrrl
Junior Member
 
Location: Indianapolis, IN

Join Date: Jul 2015
Posts: 8
Default

In my previous lab, we found that with certain plastics, the adaptors were binding to it. When doing the dilution, we were using nuclease-free water sometimes and not the suggested 10 mM Tris-HCl and that was also affecting how much the adaptors bound to the plastic. We found this out by just putting the adaptors into a plate (BioRad hardshell) and waiting for various lengths of time before running on a BioA chip. The longer it sat, the less showed up.
ScienceGrrl is offline   Reply With Quote
Old 07-17-2015, 10:13 AM   #5
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

If you don't UDG+Exo it well, it won't PCR at all. nanos has it right, ensure the ideal conditions for USER.
ECO is offline   Reply With Quote
Old 07-17-2015, 03:19 PM   #6
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

If you are starting with one microgram of DNA you shouldn't need PCR at all, much less 18 cycles. Also, at that amount of starting material differences in ligation efficiency probably don't matter much, so I also recommend going with TruSeq-style adapters so that you don't need a USER step. And you don't necessarily need to get these from Illumina, Bioo Scientific offers many Y-shaped adapter options, including PCR free, which I think would be best if you are starting with one microgram of DNA. As a disclaimer, I work for Bioo Scientific.
kerplunk412 is offline   Reply With Quote
Old 07-18-2015, 06:47 AM   #7
nanos
Member
 
Location: Vienna

Join Date: May 2010
Posts: 11
Default

If you want to go for Y-shaped adaptors, you can also just order the two strands as oligo (best is HPLC purified) and perform a simple annealing reaction before you do the ligation. We did that for a long time and it worked quite well (maybe a little less efficient than the NEB adaptors)
nanos is offline   Reply With Quote
Reply

Tags
illumina, nebnext, ngs

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 03:23 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO