[cascoamarillo]

Thanks.
I was just wondering if someone would have a little bit more percised answer.
My problem is that my fragments of interest range from 150bp up to 400bp and I need to get rid of the 120-130bp fragments. They are just so close. I tried a 1.8 :1 ratio and was successful once but starting out with less DNA I could not reporduce that.

yeah, the PAGE is very effective for that sizes (although more time consume); I did it with my small RNA libraries and apparently I get rid off all the adapter-adapter artifacts.

Maybe the system Pippin Prep gives you another solution:

http://www.sagescience.com/

[pbluescript]

Thanks.
I was just wondering if someone would have a little bit more percised answer.
My problem is that my fragments of interest range from 150bp up to 400bp and I need to get rid of the 120-130bp fragments. They are just so close. I tried a 1.8 :1 ratio and was successful once but starting out with less DNA I could not reporduce that.

Ah... that would be a problem. For getting separation resolution like that, the beads and even an agarose gel wouldn't be that great. You might want to try PAGE or, if you have the money, the Caliper XT.

http://www.caliperls.com/products/labchip-systems/labchip-xt.htm

The Caliper is capable of some very tight size separation.

[dagmar]

Read post 16 in this thread.

Thanks.
I was just wondering if someone would have a little bit more percised answer.
My problem is that my fragments of interest range from 150bp up to 400bp and I need to get rid of the 120-130bp fragments. They are just so close. I tried a 1.8 :1 ratio and was successful once but starting out with less DNA I could not reporduce that.

[bioliyezhang]

some suggestion on gel purification

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.

Hi, just one comment.
I do think the gel purification will help, if the inserted DNA size is big enough (above 100bp). And instead of using agarose gel, you can try use PAGE gel, which give you better separation. And you can just break the PAGE gel into smaller pieces and do the PCR.

[pbluescript]

Can you give us any good advice which ratio of Ampure beads and sample one should use in oder to get rid of 120-130 bp fragments.
As far as I understood the more beads you use the more bigger fragments
you capture so the ratio shold be < 1.8 : 1 correct?
Thanks!

Read post 16 in this thread.

[filippos]

Hi everyone,
last month I used the Truseq kit for RNAseq by Illumina and it worked like a charm. Previously I had to do 2-3 gel purifications in the end, but with this kit and the AMPpure beads I got a really clean picture in the end, with no adapter dimers or anything. I am new to all this, but these beads seem to do a very good job. Here is a picture of my libraries, before size selection. The sequencing worked fine and I got ~60M reads and ~92% of them were mappable to the mm9 so I guess that I got rid of most of the dimers.

[dagmar]

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.

Can you give us any good advice which ratio of Ampure beads and sample one should use in oder to get rid of 120-130 bp fragments.
As far as I understood the more beads you use the more bigger fragments
you capture so the ratio shold be < 1.8 : 1 correct?
Thanks!

[JHU-ChIPmaniac]

Hello JHU-ChIPmaniac,
I am thinking about using the E-gel as well.
I have one question to the E-gel following amplification.
What kind of PCR purification do you do before running the gel cassette?
Or have you made the experience that their is no step in between needed?
Thanks!!!

I don't usually do a PCR cleanup but if I do I just use the Qiagen PCR cleanup but this is usually just to reduce the volume so I can load everything into 1 well.

[dagmar]

Hello JHU-ChIPmaniac,
I am thinking about using the E-gel as well.
I have one question to the E-gel following amplification.
What kind of PCR purification do you do before running the gel cassette?
Or have you made the experience that their is no step in between needed?
Thanks!!!

[JHU-ChIPmaniac]

I also use the E-gel 2% size select gels following amplification. This gets rid of all the adaptor species in the libraries. I typically have plenty of library for sequencing.

[odile]

At 120bp, it is almost certainly adapter-adapter ligations. They will be sequenced by the machine and you will probably end up with lots of identical reads that match the adapter sequence.

Yes, I had tons in my first run .
I'm surprised Illumina doesn't try to improve their adaptors to avoid such issues.

[Simone78]

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.

I was interested in the double size selection and I found this paper:

http://www.ncbi.nlm.nih.gov/pubmed/20676378

unfortunately, beside the batch-to-batch variability, I was never able to get a 100% pure fraction. I always had some degree of "contamination" from shorter or longer fragments (visible at Bionalyzer, I never run them on a regular agarose gel). Therefore I chose the E-gels and I am very happy with that.

[pbluescript]

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.
Best,
Odile

At 120bp, it is almost certainly adapter-adapter ligations. They will be sequenced by the machine and you will probably end up with lots of identical reads that match the adapter sequence.

[odile]

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.
Best,
Odile

[cybeline]

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.