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Old 07-18-2011, 09:45 PM   #1
Kitty
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Smile 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!
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Old 07-19-2011, 04:44 PM   #2
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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?
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Old 07-19-2011, 06:15 PM   #3
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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
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Old 07-19-2011, 09:35 PM   #4
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Quote:
Originally Posted by ECO View Post
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!
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Old 07-19-2011, 10:10 PM   #5
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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.
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Old 07-20-2011, 01:27 PM   #6
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Can you explain your concern in more detail? It should not be a problem to prepare paired-end libraries using the standard Nextera protocol.
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Old 07-20-2011, 09:02 PM   #7
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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?
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Old 07-21-2011, 05:24 AM   #8
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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.
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Old 07-21-2011, 07:05 AM   #9
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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.
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Old 07-21-2011, 07:26 AM   #10
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Quote:
Originally Posted by epibio View Post
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

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Old 07-21-2011, 08:53 PM   #11
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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 09:04 PM.
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Old 07-21-2011, 09:01 PM   #12
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Sorry, it seems the url above doesn't work. See bioanalyzer images in the attachment.
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File Type: pdf Nextera1.pdf (226.6 KB, 137 views)
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Old 07-22-2011, 05:45 AM   #13
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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.
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Old 07-23-2011, 06:52 AM   #14
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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.

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Old 07-26-2011, 03:09 AM   #15
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I have finished the library QC by RNA 6000 chip, see pictures below. It seems there does exsit "overamplification".
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File Type: jpg P2 RNA6000.jpg (75.5 KB, 125 views)
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