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Old 04-18-2011, 08:10 AM   #1
cybeline
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Default Primer dimer problem

Hello,

I have just finished prepping multiplex samples for paired end Illumina sequencing using NEB products but after a bioanalyzer I have a lot of primer dimer at 75bp and around 120bp which seems even stronger than my bands at 300bp.
The sequencing facility does not want to sequence them due to the primer dimer problem.

Anybody else having the same problem?

I was suggested to re-gel purify all the samples but I am afraid that I will end up with not enough DNA to sequence.

Any other suggestion or the effect it could have if we sequence it like this?

Attached is a picture of the caliper data.

Thank you,
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File Type: pdf HS UCI 1-2 dil_2011-03-10_11-58-45_Gel.pdf (48.0 KB, 961 views)
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Old 04-18-2011, 09:16 AM   #2
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If you sequence it like this, you will lose a lot of data due to the machine just sequencing primer dimers.
You could size select using AMPure beads. That will give you better yield than gel extraction.
When you do your ligations next time, use less adapter and be sure to add the ligase last to your reactions, not in a master mix.
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Old 04-18-2011, 09:46 AM   #3
cybeline
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Quote:
Originally Posted by pbluescript View Post
If you sequence it like this, you will lose a lot of data due to the machine just sequencing primer dimers.
You could size select using AMPure beads. That will give you better yield than gel extraction.
When you do your ligations next time, use less adapter and be sure to add the ligase last to your reactions, not in a master mix.
Thank you very much for your answer.

I will look into Ampure beads for the cleanup.
But I believe the primer diner occurs during the pcr enrichment step since after the ligation I gel purify my samples.
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Old 04-18-2011, 09:54 AM   #4
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Sorry, I shouldn't have said primer dimers. They would be more accurately described as adapter-adapter ligations. The primers are nowhere near 120bp in length, so what you are actually amplifying are adapters that ligated to each other rather than to your sample. My suggestions for altering your ligation reaction should help with that.
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Old 04-18-2011, 09:54 AM   #5
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Hi,

One stupid question; are you sure it's primer dimer?, or maybe it could be a double strain adapter-adapter construct which is amplified after the pcr.
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Old 04-18-2011, 10:26 AM   #6
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Hi,

One stupid question; are you sure it's primer dimer?, or maybe it could be a double strain adapter-adapter construct which is amplified after the pcr.
I assume that the 75 bp peak is unreacted primer and that the 150 bp peak is a ligation artifact of two primers ligating together. But I thought it was the enrichment primers and not the ligation adapters.

The way I made my multiplex the barcodes were added with the adapters and not with the enrichment pcr.

Please let me know if that helps and thank you.
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Old 04-18-2011, 10:27 AM   #7
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Quote:
Originally Posted by pbluescript View Post
Sorry, I shouldn't have said primer dimers. They would be more accurately described as adapter-adapter ligations. The primers are nowhere near 120bp in length, so what you are actually amplifying are adapters that ligated to each other rather than to your sample. My suggestions for altering your ligation reaction should help with that.
So if I understand correctly, I should use less enrichment primers?
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Old 04-18-2011, 10:34 AM   #8
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So if I understand correctly, I should use less enrichment primers?
You should use less of the adapters that you ligated to your sample, not less enrichment primers. A 10:1 molar ratio at most, but I usually use less. If you have too many of them during your ligation reaction, they will end up ligating to each other and then that will end up amplifying in your enrichment PCR step. Even if you size selected with an agarose gel, you can still get some, especially if you used a razor to cut out bands. Since they are smaller than your sample+adapter, the adapter-only ligation products can dominate the reaction even if they are at a lower concentration than your sample to begin with.
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Old 04-18-2011, 10:38 AM   #9
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Quote:
Originally Posted by pbluescript View Post
You should use less of the adapters that you ligated to your sample, not less enrichment primers. A 10:1 molar ratio at most, but I usually use less. If you have too many of them during your ligation reaction, they will end up ligating to each other and then that will end up amplifying in your enrichment PCR step. Even if you size selected with an agarose gel, you can still get some, especially if you used a razor to cut out bands. Since they are smaller than your sample+adapter, the adapter-only ligation products can dominate the reaction even if they are at a lower concentration than your sample to begin with.
Thank you very much for your answer.

Could you please expand on the role of the razor to cut the gel? Thanks again
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Old 04-18-2011, 10:42 AM   #10
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Thank you very much for your answer.

Could you please expand on the role of the razor to cut the gel? Thanks again
Sure. The razor is usually very long compared to the region of the gel you want to cut out. It will touch lots of other sizes of DNA in the gel, which can lead to intra-sample contamination with sizes you didn't originally intend to be in your sample. I got better results when I purchased some gel cutting tips or used AMpure beads for size selection.
Taking a look at your gel, did you cut out everything between 300-400bp for samples CB5-8?
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Old 04-18-2011, 10:52 AM   #11
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Sure. The razor is usually very long compared to the region of the gel you want to cut out. It will touch lots of other sizes of DNA in the gel, which can lead to intra-sample contamination with sizes you didn't originally intend to be in your sample. I got better results when I purchased some gel cutting tips or used AMpure beads for size selection.
Taking a look at your gel, did you cut out everything between 300-400bp for samples CB5-8?
Yes. That gel is after everything, just before submitting it to sequencing.
They suggested to re-gel purify all the samples between 300 to 400. So far I have done cb1-4 but I obtained final concentration of 1 to 4 ng/ul so around 4.5 to 17 nM final concentration. I lost a lot of dna from the 2nd purification.
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Old 04-18-2011, 11:47 AM   #12
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I've had the same issue but routinely use a second gel purification to fix this. Although, you absolutely lose some DNA with the second gel purification. I use the Qiagen Minelute kit to elute with a smaller volume (10-12ul) in order to increase the concentration. This seems to do the trick nicely. I haven't played around with the AMpure beads but will be doing so soon.
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Old 04-18-2011, 11:16 PM   #13
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I had the same problem(s). To get rid of the adaptor-adaptor dimers I now do size selection using 2% E-gel (great because you can also select more than one single fraction, so you have a backup in case something goes wrong during the pre-amplification step). And you don't need an additional purification step, just pipette out the water (containing your DNA) from the well. To remove the primer dimers I use AMPure XP (1.8:1 ratio) which removes (almost) 100% of the fragments <100 bp.
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Old 04-19-2011, 05:52 AM   #14
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Those are great answers, thank you very much!

Simone78, could you please link the E-gel? and how do you get rid of the 120bp fragments?

Thanks again,

Another question, does this happen as well while using the Illumina truseq kit?
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Old 04-19-2011, 07:36 AM   #15
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2% SizeSelect E-gel from Invitrogen:

http://www.invitrogen.com/site/us/en...izeselect.html

The peak you get on the Bioanalyzer after size selection is very narrow (see attachment) and you can collect as many fractions as you want (I usually collect 4 or 5 fractions at 30 sec interval, which corresponds to about 50 bp difference).

After AMPure XP you won't probably remove 100% of the band at 120 bp, but you'll make this fraction much smaller than after Qiagen columns (for example).
Playing with the ratio DNA/beads might further improve the final results, but I haven't tried it yet.

Sorry, no experience with the truseq kit. I'm using a modified version of the PE Illumina protocol for whole methylome seq.

Last edited by Simone78; 08-24-2017 at 05:42 AM.
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Old 04-19-2011, 10:25 AM   #16
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I have attached an image showing the effect of altering the ratio of AMPure beads/sample. You can see that using a lower ratio will remove increasing sizes of DNA fragments. You can even use this for size selection by using two different concentrations. For example, if you wanted to get a band centered ~400 bp, you could use 0.7X beads and then take the supernatant. This would include all the DNA fragments that were smaller than ~500bp and didn't bind to the beads. Then mix the supernatant with 0.9X beads and everything larger than ~300bp would bind. Elute the bound DNA and you'll have a fairly broad, but good enough to sequence, library.
If you try the beads, there can be a bit of lot-to-lot variability, so I would test them with a 100 bp library as I did in this gel.

I haven't used the Truseq kit, but I have used the Truseq adapters with outsourced enzymes/buffers and the same problem can occur.
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Old 04-19-2011, 10:30 AM   #17
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Quote:
Originally Posted by pbluescript View Post
I have attached an image showing the effect of altering the ratio of AMPure beads/sample. You can see that using a lower ratio will remove increasing sizes of DNA fragments. You can even use this for size selection by using two different concentrations. For example, if you wanted to get a band centered ~400 bp, you could use 0.7X beads and then take the supernatant. This would include all the DNA fragments that were smaller than ~500bp and didn't bind to the beads. Then mix the supernatant with 0.9X beads and everything larger than ~300bp would bind. Elute the bound DNA and you'll have a fairly broad, but good enough to sequence, library.
If you try the beads, there can be a bit of lot-to-lot variability, so I would test them with a 100 bp library as I did in this gel.

I haven't used the Truseq kit, but I have used the Truseq adapters with outsourced enzymes/buffers and the same problem can occur.
This is great, thank you.

So do you only use this once after the ligation step? or also in the very end to make sure the libraries are fully cleaned?

Thanks again.
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Old 04-19-2011, 10:53 AM   #18
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It depends on the sample and what you want to sequence, but in your case you could use 1.1X or 1.2X beads to remove the adapter-adapter ligations and retain your 300-400 bp library. This would retain more DNA than doing another gel extraction.
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Old 04-19-2011, 11:30 AM   #19
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Quote:
Originally Posted by pbluescript View Post
It depends on the sample and what you want to sequence, but in your case you could use 1.1X or 1.2X beads to remove the adapter-adapter ligations and retain your 300-400 bp library. This would retain more DNA than doing another gel extraction.
Thank you.
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Old 04-19-2011, 02:59 PM   #20
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Hello everyone,

I made libraries that I sent to some lab for sequencing and also got tons of these artefacts/adaptor dimers. My problem is that I work with very degraded DNA so my library is around 150bp...too close to get rid of the 130bp artefact. Does anyone knows the sequence of the artefact? Are the dimers truly form the adaptors or could they come from P1.1 and P2.1?

I'm at loss.
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