NEBNext Adapter Ligation Efficiency

mbk0asis · 06-12-2015, 02:09 AM

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>

IdahoRAD · 07-11-2015, 01:06 PM

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?

nanos · 07-12-2015, 03:44 AM

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 37°C, purify the DNA with Ampure beads and then perform the PCR.

That clearly improved the yield of our libraries.

ScienceGrrl · 07-17-2015, 10:53 AM

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.

ECO · 07-17-2015, 11:13 AM

If you don't UDG+Exo it well, it won't PCR at all. nanos has it right, ensure the ideal conditions for USER.

kerplunk412 · 07-17-2015, 04:19 PM

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.

nanos · 07-18-2015, 07:47 AM

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)

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06-12-2015, 02:09 AM	#1
mbk0asis Member Location: Korea Join Date: Dec 2011 Posts: 41	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>

07-11-2015, 01:06 PM	#2
IdahoRAD Junior Member Location: Idaho Join Date: Jan 2014 Posts: 9	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?

07-12-2015, 03:44 AM	#3
nanos Member Location: Vienna Join Date: May 2010 Posts: 11	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 37°C, purify the DNA with Ampure beads and then perform the PCR. That clearly improved the yield of our libraries.

07-17-2015, 10:53 AM	#4
ScienceGrrl Junior Member Location: Indianapolis, IN Join Date: Jul 2015 Posts: 8	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.

07-17-2015, 11:13 AM	#5
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	If you don't UDG+Exo it well, it won't PCR at all. nanos has it right, ensure the ideal conditions for USER.

07-17-2015, 04:19 PM	#6
kerplunk412 Senior Member Location: Bioo Scientific, Austin, TX, USA Join Date: Jun 2012 Posts: 119	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.

07-18-2015, 07:47 AM	#7
nanos Member Location: Vienna Join Date: May 2010 Posts: 11	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)