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Old 04-27-2011, 12:20 PM   #41
patisev
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Default adaptor-adaptor ligation problem with SOLiD small RNA library

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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.
I seem to have the same problem while constructing small RNA libraries for Solid sequencing, do you have any experience with this equipment? Since the fragments I should select are almost the same size as adaptor-adaptor fragments, we are having trouble collecting them from the gel. Do you think that using less adaptor could help me out too? Any experience with Invitrogen eGel for this kind of size selection? Thanks!
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Old 04-27-2011, 02:06 PM   #42
captainentropy
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If you have access to one, I recommend the Pippin Prep. The lab next door to me tested the Caliper XT recently but settled on and purchased the Pippin (which we both used in another lab before). Since the Pippin uses electroelution there is little loss of DNA in the range you are selecting for compared to the manual gel-excision method - you'll lose at least 50% of your library that way in addition to the possibility of cross-contamination.

However, after the elution step there is no need for purification of the DNA prior to the PCR, you will only lose DNA doing that. You can amplify directly from the eluted sample. What I do is I take the elution volume (I elute 200-600bp) and divide it into 36 uL aliquots and how ever many that is is how many PCR reactions I set up for that sample. Usually this is between 7-12. Doing this means you are able to maximize the amount of size-selected DNA in the PCR step resulting in more library. This also means you can get away with fewer amplification cycles thus reducing the number of potential PCR artifacts.

As for the OP question, the 124 bp peak is definitely amplified adapters. I've seen it many times. The Pippin (or CaliperXT) will reduce/eliminate this. After I switched to the Pippin I've only seen any adapter peak once, and it was small compared to my size-selected library. Also, reducing the adapter dilution ratio helps. I use 1:30. However, my lab is moving away from that and we are now going to try to optimize the dilution amount for each library. More work, but we'll have better libraries. Also, Illumina (I think it was them) now suggests using longer ligation times. I do overnight ligations anyway.
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Old 04-28-2011, 04:49 PM   #43
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Hi Captainentropy,
I am glad to here you recommend the Pippin Prep. The few samples I just recently got to run were size selected very well but I still have the problem of loading my samples. Did you ever experience that the sample wells would refill with buffer before you could load your sample? Even if I would load my sample very quickly after emptying the sample well I would loose some of my sample do overflow. I do not want to take that risk but running only 2 to 3 samples on one cassette is very pricy too.
I ligate over night as well. I also tried using less adapters but this would always end up in almost no library at all. May I asked with how much DNA you start out with.
Thanks!!!
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Old 04-28-2011, 05:18 PM   #44
cybeline
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I was just looking at the TruSeq kit from Illumina. After ligation they do 2 AMpure beads purification and then a gel purification.
Seemed like overkill and I am a little concerned about loss of DNA.
Any thoughts?
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Old 04-28-2011, 07:33 PM   #45
captainentropy
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@dagmar. Yes, I've seen that happen with the well. Only once or twice maybe. It was probably just a malformed well allowing more buffer to flow into it since the buffer chamber is right next to it. If you're seeing that all the time then I'm not sure. Maybe poking a hole in the bottom of the well would cause that?

I try to use at least 100ng of ChIPed DNA if I can. If I have more then I use more. I've gone down to 1:50 dilution and got good library yield based on the Bioanalyzer trace. In fact for that one test it was slightly better than 1:30, but I'd say with low amounts of starting material 1:30 is fine. I have used 1 ug of starting DNA with 1:30 adapters and still got great library yield based on Bioanalyzer. We're going to try and optimize that step though, finding the optimal amount of adapters to add to 10, 50, 100, 500 ng, etc. of DNA.
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Old 04-29-2011, 07:32 AM   #46
dagmar
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@Captainentropy

In the first 5 test cassettes I had it happen with 2 out of 5 wells.
But with the first new cassette it happened only in one well.
I am using 2% Ethidium Bromide free cassettes and I definitely did not touch the well walls or bottom for sure. Hope this improves and I just had bad luck with the first ones.
Have you ever run a PCR product on the Pippin Prep without a purification step like columns or beads in between?

Your adapter dilution sound very promising.
I think I will try it again. Thanks!!!

If you start out with 100ng you probably are also dependent on the high sensitivity chips. Have you ever had problems running samples from the Pippin Prep on an Agilent? The elution buffer contains much more salt than they recommend for these chips.
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Old 04-29-2011, 10:02 AM   #47
pbluescript
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Quote:
Originally Posted by patisev View Post
I seem to have the same problem while constructing small RNA libraries for Solid sequencing, do you have any experience with this equipment? Since the fragments I should select are almost the same size as adaptor-adaptor fragments, we are having trouble collecting them from the gel. Do you think that using less adaptor could help me out too? Any experience with Invitrogen eGel for this kind of size selection? Thanks!
I have used a 10% TBE-Urea polyacrylamide gel from Invitrogen for this purpose before and it worked well.
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Old 04-29-2011, 10:05 AM   #48
pbluescript
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Quote:
Originally Posted by cybeline View Post
I was just looking at the TruSeq kit from Illumina. After ligation they do 2 AMpure beads purification and then a gel purification.
Seemed like overkill and I am a little concerned about loss of DNA.
Any thoughts?
My guess is that it's an attempt to solve the problem discussed in this thread, the prevalence of adapter-adapter ligation products. It will lead to loss of product, but you have to weigh that with the problems caused by putting adapter ligations on the flowcell.
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Old 04-29-2011, 10:58 AM   #49
captainentropy
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Quote:
Originally Posted by dagmar View Post
@Captainentropy

...
Have you ever run a PCR product on the Pippin Prep without a purification step like columns or beads in between?

...
If you start out with 100ng you probably are also dependent on the high sensitivity chips. Have you ever had problems running samples from the Pippin Prep on an Agilent? The elution buffer contains much more salt than they recommend for these chips.
1st question - no. I'm only using the Pippin for size selection prior to PCR.
2nd question - after the Pippin I use the eluate directly in the PCR with no problems. After that I clean them up using the Qiagen PCR cleanup kit.
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Old 05-06-2011, 02:37 PM   #50
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Quote:
Originally Posted by captainentropy View Post
However, after the elution step there is no need for purification of the DNA prior to the PCR, you will only lose DNA doing that. You can amplify directly from the eluted sample. What I do is I take the elution volume (I elute 200-600bp) and divide it into 36 uL aliquots and how ever many that is is how many PCR reactions I set up for that sample. Usually this is between 7-12. Doing this means you are able to maximize the amount of size-selected DNA in the PCR step resulting in more library. This also means you can get away with fewer amplification cycles thus reducing the number of potential PCR artifacts.
I learned a new trick today with the Pippin Prep. If you leave the tape covering the elution chamber in place you can elute in a much smaller volume, usually around 20-30 uL. This is regardless of the size range being selected.
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Old 05-08-2011, 10:15 AM   #51
monad
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Originally Posted by jgibbons1 View Post
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.
2nd gel purification is the best option here, but you lose lots of library DNA. In this case, you can set additional round of PCR will take care of it. Stick with not more than 5 round of PCR. If your bioanalyer peak is good, you will be fine even with 10-15 cycles of PCR.
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Old 05-13-2011, 12:55 PM   #52
odile
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Would anyone have a diagram with the sequence of the amplified adaptor dimer artefact?
I just don't understand how it can come up to 120-130bp.
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Old 05-13-2011, 02:02 PM   #53
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Quote:
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Would anyone have a diagram with the sequence of the amplified adaptor dimer artefact?
I just don't understand how it can come up to 120-130bp.
The paired end primers used are 58 and 61 bp long, and 58+61 = 119, so I've always thought there should be a dimer at ~120 bp but people typically say 130. I don't know why.
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Old 05-13-2011, 02:12 PM   #54
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I do single end and the adaptors are 33 and 34bp. If you add primers, it makes 58bp + 34bp + 1 (the A)= 92bp
Does everyone have the same artefact or are they different between single end and paired end?
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Old 05-16-2011, 03:21 PM   #55
genemaster
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Default Hello

I always see adapter dimer at about 123-124 bp. Given that TruSeq index adapters do add up to about 124 (62+62?) with your BioAnalyzer being a mere 1% off, these numbers are correct.
TruSeq protocol has too many cleanup steps and loss of material is inevitable. However, adapter/ PCR mix titration should take care of any imbalance in the final PCR.
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Old 05-17-2011, 06:57 AM   #56
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So artefacts with paired end and TruSeq adaptors seem to make sense, but not those obtained with single end adaptors. What am I missing?
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Old 05-31-2011, 09:20 PM   #57
Dodu
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Default Pippin Prep Max Loading

Back to the Pippin prep topic, the company claim the optimal sensitivity for separation is 10-15ng DNA band. I'm a little concerned to use Pippin to do second purification after PCR since the amount of PCR product is far above the optimal sensitivity, though below the max load limit (4ug DNA band).
I'm wondering if anyone has titrate the separation sensitivity to higher DNA amount 200-4ug band (in 200-500 range). Thanks!
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Old 06-02-2011, 02:06 PM   #58
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Default Pippin prep & amplification free libraries?

Does anyone know if it's possible to take the eluate from the pippin prep and load directly to an Illumina flowcell? I'm wondering whether it could be used to simultaneously size-select & cleanup amplification-free libraries.
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Old 06-07-2011, 01:15 PM   #59
pmiguel
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Quote:
Originally Posted by koadman View Post
Does anyone know if it's possible to take the eluate from the pippin prep and load directly to an Illumina flowcell? I'm wondering whether it could be used to simultaneously size-select & cleanup amplification-free libraries.
I have a partial answer. We tried a few modifications to the TruSeq protocol, one of which was to do the PCR step on non-size selected library, followed by size selection on the Pippin Prep. Our yield of final library was much lower than what we got following the normal protocol. But it was still enough to sequence.

It worked.

But there are a couple of additional issues for a non-amplified library:

(1) The Y-adapters apparently cause library fragments to migrate aberrantly on some, but not all, electrophoretic conditions. Details are in another thread. I think they may run at their correct size on e-gels, but appear larger than they actually are under most commonly used electrophoretic conditions. After enrichment PCR the adapters are no longer Y-ed, so the effect disappears and you see the amplicon's true sizes.

(2) Just to state the obvious: when you work with very limiting amounts of DNA, you have to watch losses to binding against plastic-ware, etc. A typical microfuge tube may be able to bind 1 ng of DNA (complete guess.) So, if you have 200 ng, who cares. But if you have 1 ng total, you might lose the majority of your library if you don't use low-bind plastic-ware.

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Old 11-03-2011, 10:11 PM   #60
molly burns
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Default want to understand science behind short fragment removal by ampure beads

I would like to understand more about double SPRI method to remove small fragments or adapters. I read that if I use 1.8 V of Ampure beads, only large DNA fragments will bind to the beads. On the other hand, if I go sequentially, that is use 0.8 V beads, it will only bind small fragments and large fragments will stay in sup. I can then recover the large fragments by adding another 1 V of beads. How is it possible then to remove small fragments by adding 1.8 V of beads? How does then small fragments stays in solu with 1.8 V of beads?
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