Illumina Pair end library protocol

Berkeley2010 · 09-14-2010, 03:07 PM

Hi,

I am looking for a protocol for pair-end library preparation. Has anybody tried making the library without using the illumina kit. Also, does anybody have information about the ligation ratio for adapters.
Any help is appreciated.

Thanks

NextGenSeq · 09-14-2010, 03:13 PM

NEB sells a paired end kit that is ~10X cheaper than Illuimina. It doesn't come with primers but the primer sequences are listed in the sticky and at:

genomics.med.tufts.edu/documents/htseq_protocol_for_illumina_paired.pdf

Berkeley2010 · 09-14-2010, 03:34 PM

Thanks NextGenSeq. I will check that out.

SeqR&D · 09-15-2010, 03:23 PM

Adapter to library ratio is around 10:1

csquared · 09-23-2010, 08:42 AM

The Tufts protocol is nicely detailed but one word of caution: The lack of purification following the end-repair step means that there are free dNTPs in the subsequent Add-A reaction. Although the additional dATP added during Add-A is almost 100-fold excess compared to G, T, and C, there is an assumption being made that the Add-A step will be highly biased towards only using the abundant A. Probably a reasonable assumption but use at your own risk.

gogreen · 10-04-2010, 02:20 AM

We are currently using the NEB master mix kit for the illumina sample prep kit, I've prepared quite a lot of libraries with the kit and satisfied with the quality and ease of usage because it saves a lot of pipetting (hence, possible errors) compared to the normal kit. As NextGenSeq pointed out, it is almost 10X less expensive compared to the Illumina kit.

Berkeley2010 · 10-05-2010, 08:52 AM

Thanks everybody. So, I finally decided to go ahead and order adapter and primer sequences listed in tuft's protocol. Did anybody try to use these sequences from tuft's protocol. They have locked nucleotide sequence and not the phosphothioated bond.

BTS · 10-05-2010, 01:00 PM

I was recently forwarded a link about Bioo Scientific.

http://www.newswiretoday.com/news/78473/

It looks like their kits are even cheaper than NEB's. Does anyone know anything about these kits? We are just getting started with a GAIIx and if this is going to be a better/cheaper option than NEB we are going to make a switch. Thanks!

saikumarkv · 10-07-2010, 12:42 PM

I talked to Kip @ Tufts sequencing core who is extremely helpful (thanks Kip) (http://genomics.med.tufts.edu/home/sampleprep), regarding their adapters. They now switched to phosphorothioate bond modification at the last T on OLJ131.

advanT · 10-11-2010, 07:06 PM

We use these kits in all our DNA library preparations. Very happy with them.

Quote:

Originally Posted by BTS

I was recently forwarded a link about Bioo Scientific.

http://www.newswiretoday.com/news/78473/

It looks like their kits are even cheaper than NEB's. Does anyone know anything about these kits? We are just getting started with a GAIIx and if this is going to be a better/cheaper option than NEB we are going to make a switch. Thanks!

BTS · 10-12-2010, 05:17 AM

Thanks for the info advanT!

Berkeley2010 · 10-21-2010, 11:23 AM

Does anyone know if I can use Gelstar Nucleic acid stain (syber green) to stain my gel for the size selection step. Also, is E-gel better than running a homemade 2% agarose gel?

jgibbons1 · 10-21-2010, 12:14 PM

We've used syber green instead of EtBr and it's fine. Also, we routinely run 2.5% agarose gels for size selection and they are also fine.

saikumarkv · 10-21-2010, 02:05 PM

I just finished comparing the NEBNext Reagent set 1 and Illumina’s paired end sample prep kit using different amounts of ChIP input DNA (1-10 ng). I used 2 different dilutions of Illumina’s PE adapter oligos (1/10-50nM final and 1/30-16.7nM final) for 10ng. I made dilutions proportional to different amounts of starting DNA amounts. In my hands NEB worked way better. I got clean single peaks with no contaminating primer dimer or higher molecular weight humps. Using 1/30 dilution of the adapters gave the cleanest peaks. Even with 1ng I have about 35ul of 21.5 nM DNA. Illumina PE prep kit had a little bit of a hump @ 150-200 range. I’m doing the qPCR quantification using the Kappa kit. I have no idea how to insert the images of Bioanalyzer traces.

saikumarkv · 10-26-2010, 11:34 AM

Just finished estimating my library prep using the KAPPA qPCR kit. I'm actually estimating more of the library than by Qubit measurement. Using 1 ng of ChIP input DNA I'm getting 37nM by Qubit and 53nM by qPCR. For 5 ng starting amounts, 45nm by Qubit and 65nM by qPCR. I'm starting my real experiment with 32 ChIP samples.

Berkeley2010 · 11-12-2010, 03:11 PM

Hi,

I am trying to enrich my pair-end library. I made two libraries 300 bp and 600 bp. At the end PCR step, I get only 26 ng/ul and 15ng/ul for the libraries. This was estimated by nanodrop. I used 10 PCR cycles. How can I get more library. Do I have problems with my adapters that they are not ligated properly. Any help is really appreciated.

huguesparri · 11-18-2010, 07:53 AM

If your Nanodrop concentration values are acurate, you have enough material to perform a sequencing on many complete Flow Cells for each library.

Nevertheless, I would run both libraries using a DNA1000 Chip on a Bioanalyzer to be sure of the concentration. In our experience, Nanodrop tends to over-estimate concentration values for illumina's libraries.

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09-14-2010, 03:07 PM	#1
Berkeley2010 Junior Member Location: NYC Join Date: Aug 2010 Posts: 7	Illumina Pair end library protocol Hi, I am looking for a protocol for pair-end library preparation. Has anybody tried making the library without using the illumina kit. Also, does anybody have information about the ligation ratio for adapters. Any help is appreciated. Thanks

09-14-2010, 03:13 PM	#2
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	NEB sells a paired end kit that is ~10X cheaper than Illuimina. It doesn't come with primers but the primer sequences are listed in the sticky and at: genomics.med.tufts.edu/documents/htseq_protocol_for_illumina_paired.pdf

09-14-2010, 03:34 PM	#3
Berkeley2010 Junior Member Location: NYC Join Date: Aug 2010 Posts: 7	Thanks NextGenSeq. I will check that out.

09-15-2010, 03:23 PM	#4
SeqR&D Member Location: San Diego Join Date: Sep 2010 Posts: 26	Adapter to library ratio is around 10:1

09-23-2010, 08:42 AM	#5
csquared Member Location: Huntsville, AL Join Date: May 2008 Posts: 67	The Tufts protocol is nicely detailed but one word of caution: The lack of purification following the end-repair step means that there are free dNTPs in the subsequent Add-A reaction. Although the additional dATP added during Add-A is almost 100-fold excess compared to G, T, and C, there is an assumption being made that the Add-A step will be highly biased towards only using the abundant A. Probably a reasonable assumption but use at your own risk.

10-04-2010, 02:20 AM	#6
gogreen Member Location: Europe Join Date: Apr 2009 Posts: 18	We are currently using the NEB master mix kit for the illumina sample prep kit, I've prepared quite a lot of libraries with the kit and satisfied with the quality and ease of usage because it saves a lot of pipetting (hence, possible errors) compared to the normal kit. As NextGenSeq pointed out, it is almost 10X less expensive compared to the Illumina kit.

10-05-2010, 08:52 AM	#7
Berkeley2010 Junior Member Location: NYC Join Date: Aug 2010 Posts: 7	Thanks everybody. So, I finally decided to go ahead and order adapter and primer sequences listed in tuft's protocol. Did anybody try to use these sequences from tuft's protocol. They have locked nucleotide sequence and not the phosphothioated bond.

10-05-2010, 01:00 PM	#8
BTS Member Location: Wisconsin Join Date: Sep 2010 Posts: 19	I was recently forwarded a link about Bioo Scientific. http://www.newswiretoday.com/news/78473/ It looks like their kits are even cheaper than NEB's. Does anyone know anything about these kits? We are just getting started with a GAIIx and if this is going to be a better/cheaper option than NEB we are going to make a switch. Thanks!

10-07-2010, 12:42 PM	#9
saikumarkv Member Location: Cincinnati Join Date: Dec 2009 Posts: 11	I talked to Kip @ Tufts sequencing core who is extremely helpful (thanks Kip) (http://genomics.med.tufts.edu/home/sampleprep), regarding their adapters. They now switched to phosphorothioate bond modification at the last T on OLJ131.

10-12-2010, 05:17 AM	#11
BTS Member Location: Wisconsin Join Date: Sep 2010 Posts: 19	Thanks for the info advanT!

10-21-2010, 11:23 AM	#12
Berkeley2010 Junior Member Location: NYC Join Date: Aug 2010 Posts: 7	Does anyone know if I can use Gelstar Nucleic acid stain (syber green) to stain my gel for the size selection step. Also, is E-gel better than running a homemade 2% agarose gel?

10-21-2010, 12:14 PM	#13
jgibbons1 Senior Member Location: Worcester, MA Join Date: Oct 2009 Posts: 133	We've used syber green instead of EtBr and it's fine. Also, we routinely run 2.5% agarose gels for size selection and they are also fine.

10-21-2010, 02:05 PM	#14
saikumarkv Member Location: Cincinnati Join Date: Dec 2009 Posts: 11	I just finished comparing the NEBNext Reagent set 1 and Illumina’s paired end sample prep kit using different amounts of ChIP input DNA (1-10 ng). I used 2 different dilutions of Illumina’s PE adapter oligos (1/10-50nM final and 1/30-16.7nM final) for 10ng. I made dilutions proportional to different amounts of starting DNA amounts. In my hands NEB worked way better. I got clean single peaks with no contaminating primer dimer or higher molecular weight humps. Using 1/30 dilution of the adapters gave the cleanest peaks. Even with 1ng I have about 35ul of 21.5 nM DNA. Illumina PE prep kit had a little bit of a hump @ 150-200 range. I’m doing the qPCR quantification using the Kappa kit. I have no idea how to insert the images of Bioanalyzer traces.

10-26-2010, 11:34 AM	#15
saikumarkv Member Location: Cincinnati Join Date: Dec 2009 Posts: 11	Just finished estimating my library prep using the KAPPA qPCR kit. I'm actually estimating more of the library than by Qubit measurement. Using 1 ng of ChIP input DNA I'm getting 37nM by Qubit and 53nM by qPCR. For 5 ng starting amounts, 45nm by Qubit and 65nM by qPCR. I'm starting my real experiment with 32 ChIP samples.

11-12-2010, 03:11 PM	#16
Berkeley2010 Junior Member Location: NYC Join Date: Aug 2010 Posts: 7	Hi, I am trying to enrich my pair-end library. I made two libraries 300 bp and 600 bp. At the end PCR step, I get only 26 ng/ul and 15ng/ul for the libraries. This was estimated by nanodrop. I used 10 PCR cycles. How can I get more library. Do I have problems with my adapters that they are not ligated properly. Any help is really appreciated.

11-18-2010, 07:53 AM	#17
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	If your Nanodrop concentration values are acurate, you have enough material to perform a sequencing on many complete Flow Cells for each library. Nevertheless, I would run both libraries using a DNA1000 Chip on a Bioanalyzer to be sure of the concentration. In our experience, Nanodrop tends to over-estimate concentration values for illumina's libraries.