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Old 04-19-2011, 06:56 PM   #21
pbluescript
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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.
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.
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Old 04-19-2011, 11:37 PM   #22
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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.
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.
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Old 04-20-2011, 07:31 AM   #23
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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.
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Old 04-20-2011, 11:17 AM   #24
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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.
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Old 04-21-2011, 08:44 AM   #25
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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!!!
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Old 04-21-2011, 08:51 AM   #26
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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.
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Old 04-21-2011, 08:53 AM   #27
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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.
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!
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Old 04-21-2011, 09:10 AM   #28
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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.
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File Type: jpg rna seq 02282011.jpg (17.0 KB, 242 views)
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Old 04-21-2011, 09:13 AM   #29
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Originally Posted by dagmar View Post
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.
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Old 04-21-2011, 09:31 AM   #30
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Default some suggestion on gel purification

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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.
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.
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Old 04-22-2011, 06:54 AM   #31
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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.
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Old 04-22-2011, 07:04 AM   #32
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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/la...labchip-xt.htm
The Caliper is capable of some very tight size separation.
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Old 04-22-2011, 07:25 AM   #33
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Originally Posted by dagmar View Post
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/
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Old 04-22-2011, 04:56 PM   #34
dagmar
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Originally Posted by pbluescript View Post
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/la...labchip-xt.htm
The Caliper is capable of some very tight size separation.
Quote:
Originally Posted by cascoamarillo View Post
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/
Thanks. I guess you just saved me time and effort.
I was planning on using the Pippin Prep next but the test runs were a little disappointing. I had problems loading samples because 2 out of 5 wells would refill with buffer and the yield from the ones that worked was very little.
Do you have any experience with the Pippin Prep? I will not get the chance to try the Caliper XP. I guess I will give the Pippin Prep a second chance so any further advice would be great.
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Old 04-26-2011, 06:50 AM   #35
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Thanks. I guess you just saved me time and effort.
I was planning on using the Pippin Prep next but the test runs were a little disappointing. I had problems loading samples because 2 out of 5 wells would refill with buffer and the yield from the ones that worked was very little.
Do you have any experience with the Pippin Prep? I will not get the chance to try the Caliper XP. I guess I will give the Pippin Prep a second chance so any further advice would be great.
The Pippin Prep that we have is been used by other people in the department, not me. They also do RNA-seq, but I'm not sure what's the potencial separation-resolution of that machine (it's still a agarose separation system). For my stuff, where the adapters artifacts (75 bp) and the library (100 bp) are so closed, was not an option.
Hope it helps.
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Old 04-26-2011, 06:56 AM   #36
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One alternative from the E-gel is the recovery gels from Lonza (flashgels). I tested them last week and they work great.
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Old 04-26-2011, 07:06 AM   #37
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to dagmar. Some different ratios are 1:1 for getting rid of 150bp and smaller. That might be useful if you don't mind taking a potential hit on the 150bp area of your library range. Alternatively 1.5:1. I'm not precisely sure of the size but it might get rid of some or all of your 120-130bp fragments.
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Old 04-27-2011, 06:47 AM   #38
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to dagmar. Some different ratios are 1:1 for getting rid of 150bp and smaller. That might be useful if you don't mind taking a potential hit on the 150bp area of your library range. Alternatively 1.5:1. I'm not precisely sure of the size but it might get rid of some or all of your 120-130bp fragments.
Thanks. I tried it out now. 1.5 which did a good job. 1.3 and 1.0 somehow did not get rid of any of the artifacts around 120bp.
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Old 04-27-2011, 06:55 AM   #39
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Originally Posted by cascoamarillo View Post
The Pippin Prep that we have is been used by other people in the department, not me. They also do RNA-seq, but I'm not sure what's the potencial separation-resolution of that machine (it's still a agarose separation system). For my stuff, where the adapters artifacts (75 bp) and the library (100 bp) are so closed, was not an option.
Hope it helps.
I finally have some results from the Pippin Prep 2% and it does seperate the 120bp artifacts from my 150bp fragments so I could imagine a 3% cassette might work for you too, but the yield is not as good as I excpected so far.
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Old 04-27-2011, 07:02 AM   #40
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One alternative from the E-gel is the recovery gels from Lonza (flashgels). I tested them last week and they work great.
Never heard of it. Sounds great. It really might be an alternative.
Can you do tight seperations like 120bp from 150bp and collect library ranges up to 300bp with it? On the Pippin prep low single nanogramms of DNA can be loeaded what about the minimum load of DNA of the Flashgels.
Thanks!
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