Nextera Problem: Single adapters in PE500 library??

Kitty · 07-18-2011, 10:45 PM

Hi, I'm a new member here.
Are there anyone use the Nextera transposase for Illumina library construction? The single-tube reaction in 2 hours seems attractive!

Kitty · 07-19-2011, 05:44 PM

In fact, I'm now trying to prepare a PE500 library using the Nextera DNA Prep Kit. However, it seems that the DNA fragment in the first step tend to link with each other as the existence of single-end adaptors at both ends. I want to know is there some good ideas towards this issue?

ECO · 07-19-2011, 07:15 PM

Kitty,

Welcome. In the future, please search first either using the forum software search function or by appending "site:seqanswers.com" to your Google query.

See this link for many discussions about Nextera. http://www.google.com/search?sourcei...seqanswers.com

Kitty · 07-19-2011, 10:35 PM

Quote:

Originally Posted by ECO

Kitty,

Welcome. In the future, please search first either using the forum software search function or by appending "site:seqanswers.com" to your Google query.

See this link for many discussions about Nextera. http://www.google.com/search?sourcei...seqanswers.com

Thanks a lot!

ECO · 07-19-2011, 11:10 PM

Ok, second (and hopefully last) etiquette piece of advice. Now that I've pointed out old threads about Nextera, it's not cool to post your question in all of them. I've deleted all of those posts.

I'm also changing this thread title to a useful one that clearly states your question. Hopefully this will garner the responses you desire.

epibio · 07-20-2011, 02:27 PM

Can you explain your concern in more detail? It should not be a problem to prepare paired-end libraries using the standard Nextera protocol.

Kitty · 07-20-2011, 10:02 PM

Ok. In brief, we analyzed the intermediate product by Agilent 2100 Bioanalyser before and after the PCR. However, it seemed that the fragments distributed around 200bp before PCR, but turned to 400~1500bp after PCR. We worried that there might be some tagment concatamers in the product. What do you think of this?

pmiguel · 07-21-2011, 06:24 AM

How many cycles of PCR did you do?

Would you post your bioanalyzer image (electropherogram)?

I don't know whether Nextera treatment resolves to a TruSeq library amplicon. But, if so, PCR of TruSeq amplicons can generate annealed "bubble" or "daisy chain" multimers that resolve into monomers upon denaturation.
--
Phillip

epibio · 07-21-2011, 08:05 AM

Most likely, you're experiencing a phenomenon called "bird-nesting" (see our blog post). As pmiguel said, this is not a problem since the denaturation step during bridge PCR will take care of it.

pmiguel · 07-21-2011, 08:26 AM

Quote:

Originally Posted by epibio

Most likely, you're experiencing a phenomenon called "bird-nesting" (see our blog post). As pmiguel said, this is not a problem since the denaturation step during bridge PCR will take care of it.

To assay without sequencing you can denature the an aliquot of the library and run it on a pico chip. See:

http://seqanswers.com/forums/showthread.php?t=12523

Not a perfect solution because it is not clear if the DNA will renature to some extent during chip loading, etc. And dsDNA runs faster than expected on an RNA bioanalyzer chip. See:

http://seqanswers.com/forums/showthread.php?t=12501

--
Phillip

Kitty · 07-21-2011, 09:53 PM

Hi Philip,

Thanks for your smart suggestions! In fact, I did do PCR for 15 cycles, and maybe that’s what does really matter (see the bioanalyzer image). While we still worry that reducing the number of PCR cycles would lead to low production (50ng starting human gDNAs).

Your idea of "Using RNA 6000 pico chips for final library QC” sounds fascinating, and I’ll try this right away to track the problems.

However, that will change to another problem: How to control the fragment sizes of Nextera transposase reaction? We need to construct many PE500 libraries from low input DNA. Is there anyone have similar experiences?

Kitty

Kitty · 07-21-2011, 10:01 PM

Sorry, it seems the url above doesn't work. See bioanalyzer images in the attachment.

epibio · 07-22-2011, 06:45 AM

15 cycles of PCR is overkill. The Nextera protocol is optimized for 50 ng of input DNA. If you follow the protocol exactly, you shouldn't have any problems with the fragment size distribution. Also, make sure you use the Nextera PCR enzyme as per the protocol.

pmiguel · 07-23-2011, 07:52 AM

Still, I think the high molecular weight fragments result from "bird nesting" (or "daisy chaining" or "bubble amplicons"). Which may mean the library is okay after denaturation.

Although true linear multimers may result after too much PCR. So running the sample denatured would be the only way I can think of to tell without doing a sequencing run.

--
Phillip

Kitty · 07-26-2011, 04:09 AM

I have finished the library QC by RNA 6000 chip, see pictures below. It seems there does exsit "overamplification".

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
TruSeq or Nextera kit for library prep?	rooty	Sample Prep / Library Generation	12	10-29-2013 07:02 AM
Nextera vs Illumina Library Prep	maggie	Illumina/Solexa	6	03-01-2012 07:01 PM
qPCR to validate/quantify Nextera library	mikmz	Sample Prep / Library Generation	0	12-14-2011 01:26 AM
Can I use same adapters for PE and single read?	ein_io	Illumina/Solexa	4	11-20-2011 01:24 PM
Nextera vs Illumina Library Prep	maggie	Illumina/Solexa	2	12-30-2010 10:58 AM

07-18-2011, 10:45 PM	#1
Kitty Member Location: China Join Date: Jul 2011 Posts: 10	Nextera Problem: Single adapters in PE500 library?? Hi, I'm a new member here. Are there anyone use the Nextera transposase for Illumina library construction? The single-tube reaction in 2 hours seems attractive!

07-21-2011, 09:53 PM	#11
Kitty Member Location: China Join Date: Jul 2011 Posts: 10	Hi Philip, Thanks for your smart suggestions! In fact, I did do PCR for 15 cycles, and maybe that’s what does really matter (see the bioanalyzer image). While we still worry that reducing the number of PCR cycles would lead to low production (50ng starting human gDNAs). Your idea of "Using RNA 6000 pico chips for final library QC” sounds fascinating, and I’ll try this right away to track the problems. However, that will change to another problem: How to control the fragment sizes of Nextera transposase reaction? We need to construct many PE500 libraries from low input DNA. Is there anyone have similar experiences? Kitty Last edited by Kitty; 07-21-2011 at 10:04 PM.

07-19-2011, 05:44 PM	#2
Kitty Member Location: China Join Date: Jul 2011 Posts: 10	In fact, I'm now trying to prepare a PE500 library using the Nextera DNA Prep Kit. However, it seems that the DNA fragment in the first step tend to link with each other as the existence of single-end adaptors at both ends. I want to know is there some good ideas towards this issue?

07-19-2011, 07:15 PM	#3
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Kitty, Welcome. In the future, please search first either using the forum software search function or by appending "site:seqanswers.com" to your Google query. See this link for many discussions about Nextera. http://www.google.com/search?sourcei...seqanswers.com

07-19-2011, 11:10 PM	#5
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Ok, second (and hopefully last) etiquette piece of advice. Now that I've pointed out old threads about Nextera, it's not cool to post your question in all of them. I've deleted all of those posts. I'm also changing this thread title to a useful one that clearly states your question. Hopefully this will garner the responses you desire.

07-20-2011, 02:27 PM	#6
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	Can you explain your concern in more detail? It should not be a problem to prepare paired-end libraries using the standard Nextera protocol.

07-20-2011, 10:02 PM	#7
Kitty Member Location: China Join Date: Jul 2011 Posts: 10	Ok. In brief, we analyzed the intermediate product by Agilent 2100 Bioanalyser before and after the PCR. However, it seemed that the fragments distributed around 200bp before PCR, but turned to 400~1500bp after PCR. We worried that there might be some tagment concatamers in the product. What do you think of this?

07-21-2011, 06:24 AM	#8
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	How many cycles of PCR did you do? Would you post your bioanalyzer image (electropherogram)? I don't know whether Nextera treatment resolves to a TruSeq library amplicon. But, if so, PCR of TruSeq amplicons can generate annealed "bubble" or "daisy chain" multimers that resolve into monomers upon denaturation. -- Phillip

07-21-2011, 08:05 AM	#9
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	Most likely, you're experiencing a phenomenon called "bird-nesting" (see our blog post). As pmiguel said, this is not a problem since the denaturation step during bridge PCR will take care of it.

07-22-2011, 06:45 AM	#13
epibio Registered Vendor Location: Madison, WI Join Date: May 2010 Posts: 89	15 cycles of PCR is overkill. The Nextera protocol is optimized for 50 ng of input DNA. If you follow the protocol exactly, you shouldn't have any problems with the fragment size distribution. Also, make sure you use the Nextera PCR enzyme as per the protocol.

07-23-2011, 07:52 AM	#14
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Still, I think the high molecular weight fragments result from "bird nesting" (or "daisy chaining" or "bubble amplicons"). Which may mean the library is okay after denaturation. Although true linear multimers may result after too much PCR. So running the sample denatured would be the only way I can think of to tell without doing a sequencing run. -- Phillip