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Old 02-08-2012, 11:33 AM   #1
MLog
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Question RNA-seq libraries - double peak after size selection

Hi everyone,
I get some weird results when measuring my RNA-seq libraries on Bioanalyzer and now desperately need an advice...
The libraries look like a peak of expected length (300-400 or 400-500) and a higher peak (>1000 bp according to BA) (the image is attached).
I'm using TruSeq RNA v.2 kit, following the protocol (except for using qiagen columns instead of ampure beads and a size-selection step after PCR enrichment).
This is the first time that I'm making RNA-seq libraries but I have some experience with standard DNA libraries - and none of them looked like this. It was always a peak of expected length, sometimes with traces of adapter dimers at 120 bp - but never any peaks higher than expected.
What puzzles me the most is that I got this AFTER size selection. I'm pretty sure that I cut the band about 300-500 bp and certainly not 1500-2000 bp! So this should be something generated from my 300-500 bp fragments. What could it be? A single-stranded DNA? If so, will it affect the sequencing resuts? Does it make sense to try to run these libraries or it would be safer to redo them?
Thank you very much in advance!
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File Type: jpg 8-2-12_RNAseq.jpg (61.8 KB, 518 views)

Last edited by MLog; 02-08-2012 at 01:46 PM.
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Old 02-08-2012, 01:19 PM   #2
ssing
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Hi there,

I got the exact same pattern you describe (couldn't see the image). See this post for what I found, what others suggested, and to see the end results. Let me know if you'd like more information.

http://seqanswers.com/forums/showthread.php?t=15224

Best of luck!
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Old 02-08-2012, 02:16 PM   #3
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Quote:
Originally Posted by ssing View Post
Hi there,

I got the exact same pattern you describe (couldn't see the image). See this post for what I found, what others suggested, and to see the end results. Let me know if you'd like more information.

http://seqanswers.com/forums/showthread.php?t=15224

Best of luck!
Thanks, ssing! This is a very useful link. In my case the reason is not the beads - just because I don't use them. So I will certainly try heating and cooling.
PS: there was indeed a problem with the attached image but now it should work.
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Old 02-09-2012, 04:25 AM   #4
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It is due to too many cycles of PCR. When you have a high concentration of DNA fragments with complimentary ends, they can hybridize with each other, leading to the longer fragment sizes. Did you amplify each library with the same number of cycles or did you titrate the cycle number?
If you titrate the number of PCR cycles, you can be sure you don't get these effects and introduce minimal PCR bias into your samples.
You can see the gel I have attached to this post for an example of the appearance of these double sized bands as the number of PCR cycles goes from 6-18.

Edit:
The gel is 1.5% agarose post-stained with EtBr. The ladder is an Invitrogen 1kb+ ladder, so the sizes visible are 100,200,300,400,500,650.
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Last edited by pbluescript; 03-05-2012 at 03:59 AM. Reason: Added more information about the gel.
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Old 02-12-2012, 07:49 AM   #5
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Quote:
Originally Posted by ssing View Post
Hi there,

I got the exact same pattern you describe (couldn't see the image). See this post for what I found, what others suggested, and to see the end results. Let me know if you'd like more information.

http://seqanswers.com/forums/showthread.php?t=15224

Best of luck!
Hi ssing,
Glad that worked for you. However, about the gel you ran after heating/cooling -- was that agarose? If so, did you pre-stain (eg, EtBr in loading dye and/or gel and/or running buffer) or post-stain the gel?

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Old 02-12-2012, 07:54 AM   #6
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Originally Posted by pbluescript View Post
You can see the gel I have attached to this post for an example of the appearance of these double sized bands as the number of PCR cycles goes from 6-18.
Hi pbluescript,
That is a great picture. Could we get info about the sieving matrix (agarose or acrylamide?) and whether you pre or post-stained it? And what the stain was (EtBr, SyBR-safe, SyBr-gold, etc)?

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Old 02-12-2012, 08:05 AM   #7
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Hi pbluescript,
That is a great picture. Could we get info about the sieving matrix (agarose or acrylamide?) and whether you pre or post-stained it? And what the stain was (EtBr, SyBR-safe, SyBr-gold, etc)?

--
Phillip
Sure. This one was 1.5% agarose post-stained with EtBr. I've actually tried it with 1-2% agarose post-stained with EtBr, Sybr-safe, and Sybr-gold and get identical results. I have also used the Invitrogen E-gels and see the same pattern.

I generally take an aliquot of each library pre-PCR and titrate the number of cycles to decide how many each library needs.
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Old 02-12-2012, 08:44 AM   #8
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Sure. This one was 1.5% agarose post-stained with EtBr. I've actually tried it with 1-2% agarose post-stained with EtBr, Sybr-safe, and Sybr-gold and get identical results. I have also used the Invitrogen E-gels and see the same pattern.

I generally take an aliquot of each library pre-PCR and titrate the number of cycles to decide how many each library needs.
Then I am not sure what to think. I thought most of the double peak phenomenon was the result of the extra sieving resistance BioAnalyzer chip polymer had for ssDNA vs. dsDNA. But you are showing the same phenomenon in agarose -- agarose without run-time staining. Strange.

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Old 02-13-2012, 08:18 AM   #9
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Hi Phillip,

It was a pre-stained agarose gel. Thanks!

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Old 02-14-2012, 02:41 AM   #10
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Originally Posted by pmiguel View Post
Then I am not sure what to think. I thought most of the double peak phenomenon was the result of the extra sieving resistance BioAnalyzer chip polymer had for ssDNA vs. dsDNA. But you are showing the same phenomenon in agarose -- agarose without run-time staining. Strange.

--
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I always thought it was the complimentary adapter ends from separate molecules hybridizing, increasing the overall length of the fragments. As the concentration goes up, this becomes more likely. I haven't directly tested that though.
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Old 02-14-2012, 03:25 AM   #11
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Originally Posted by pbluescript View Post
It is due to too many cycles of PCR. When you have a high concentration of DNA fragments with complimentary ends, they can hybridize with each other, leading to the longer fragment sizes. Did you amplify each library with the same number of cycles or did you titrate the cycle number?
If you titrate the number of PCR cycles, you can be sure you don't get these effects and introduce minimal PCR bias into your samples.
You can see the gel I have attached to this post for an example of the appearance of these double sized bands as the number of PCR cycles goes from 6-18.
They should pose no problem during sequencing as you denature before you load onto the cBot.
You could perform and additional PCR cycle with extra primers and mastermix if you want to check. The additional peak should disappear.
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Old 02-14-2012, 03:29 AM   #12
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They should pose no problem during sequencing as you denature before you load onto the cBot.
You could perform and additional PCR cycle with extra primers and mastermix if you want to check. The additional peak should disappear.
I agree. They've never caused me an issue. I use the appearance of the bands more as a guide to the maximum number of PCR cycles I need for a library.
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Old 02-14-2012, 02:12 PM   #13
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Hi All

This will be my first post…also first time attempting the TruSeq protocol (followed exactly - 4ug total RNA, AmPureXP beads, 15cycle PCR amp). I seemed to have gotten exactly the same result as ssing did (http://seqanswers.com/forums/showthread.php?t=15224), strong band at ±300-500bp with a HMW smear going all the way to the wells (1% EtBr pre-stained agarose gel attached). I had a heart attack when I saw this earlier today, but according to the post I have seen, it seems to be a regular result with the Truseq kit. I will try pmiguel’s advice on denaturing the libraries and rerunning the gel just to see if the smear clears up.

It seems then that these libraries are over amplified (if I understand correctly), but what effect will this have on a transcriptome assembling project? I am interested in read counts/ expression levels. Will an over amplified library result in too much of a bias in the results or will it be fine to continue with what I have?

I will still size-select for what its worth. What is a good insert size to aim for when attempting a de novo assembly?

Thanks for any advice
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Old 02-15-2012, 07:53 AM   #14
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Quote:
Originally Posted by monkey_SEQ View Post
Hi All

This will be my first post…also first time attempting the TruSeq protocol (followed exactly - 4ug total RNA, AmPureXP beads, 15cycle PCR amp). I seemed to have gotten exactly the same result as ssing did (http://seqanswers.com/forums/showthread.php?t=15224), strong band at ±300-500bp with a HMW smear going all the way to the wells (1% EtBr pre-stained agarose gel attached). I had a heart attack when I saw this earlier today, but according to the post I have seen, it seems to be a regular result with the Truseq kit. I will try pmiguel’s advice on denaturing the libraries and rerunning the gel just to see if the smear clears up.

It seems then that these libraries are over amplified (if I understand correctly), but what effect will this have on a transcriptome assembling project? I am interested in read counts/ expression levels. Will an over amplified library result in too much of a bias in the results or will it be fine to continue with what I have?

I will still size-select for what its worth. What is a good insert size to aim for when attempting a de novo assembly?

Thanks for any advice
Actually, those look fine to me. The smear is probably due to the high concentration of DNA.
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Old 03-04-2012, 07:11 PM   #15
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Originally Posted by pbluescript View Post
It is due to too many cycles of PCR. When you have a high concentration of DNA fragments with complimentary ends, they can hybridize with each other, leading to the longer fragment sizes. Did you amplify each library with the same number of cycles or did you titrate the cycle number?
If you titrate the number of PCR cycles, you can be sure you don't get these effects and introduce minimal PCR bias into your samples.
You can see the gel I have attached to this post for an example of the appearance of these double sized bands as the number of PCR cycles goes from 6-18.
Very nice. Could you please provide ladder size? Thanks!
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Old 03-05-2012, 12:49 AM   #16
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This problem comes up over and over again. There are a lot of posts on it. Illumina seems to be clueless as how to address the issue (if they had given me that job I applied for, think about all the customer's headaches that could have been avoided). Bottom line is you really need to accurately determine the optimum number of PCR cycles you need to do for each and every sample. It is a trivial amount of time in the scheme of things and will reduce PCR bias. I outline an easy way to do it here:
http://ethanomics.wordpress.com/ngs-...tion-protocol/
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Old 03-05-2012, 01:57 AM   #17
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Thanks for the reply Ethan. I will definitely implement your protocol in the future to test for optimum number of cycles of other samples. Unfortunately for these libraries I have no more tissue/ RNA left and I will only be able to get more in about 6 months or so. I wish Illumina would give better guidelines in their protocols so that newbies like me don’t just stupidly follow the protocol exactly only to realize afterwards that there are actually problems with it!!

So I size-selected a 300bp fragment (for what it was worth) and already sent it away for sequencing. I want to assemble a de novo transcriptome and then compare expression levels of different genes. In your experience, how much of an influence will an over-amplified library have on this experiment? Do you think I will be able to use the data at all?
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Old 03-05-2012, 02:25 AM   #18
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As long as the sequencing goes well, the data should be fine. The biggest problem is the stress you went though wonder what went wrong and fearing that all that time you spent preparing your samples is down the drain. But it's not a big deal, which is probably why Illumna ignores it, but they should at least mention something in their kit instructions.
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Old 03-05-2012, 02:31 AM   #19
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Sigh, that's wonderful news And yes I think I might have a stomach ulcer by now!
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Old 03-05-2012, 03:48 AM   #20
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Quote:
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Very nice. Could you please provide ladder size? Thanks!
Sure. It is an Invitrogen 1kb+ ladder, so the sizes visible are 100,200,300,400,500,650.
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