AMPure, amplicons and GS Jr enrichment beads

[suefo]

We tried for the first time using DNA (amplicons) previously purified with AMPure XP and then emPCR at 0.25 cpb with the GS FLX kits with the GS Junior emPCR kits. Our enrichment went from 15% to 40-50% (ran duplicates). Has anyone else used the GS Junior emPCR kits and do you have any recommendations. We are going to try again using .1 cpb.

Also any feedback on quality of library from amplicons using AMPure? We are getting a high percentage of dot+mixed as well as short. Roche thinks it may be the library source.

[pmiguel]

The metric "cpb", "copies per bead" requires an accurate measurement of DNA quantity and amplicon size. What method are you using to measure these?

I think what Roche means by "library source" is that the AMPure XP may not have completely removed PCR primer dimers.

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Phillip

[suefo]

I am wondering about the primer dimers too. And if perhaps another round of purification is needed or another purification process would be better than the AMPure.

I did not perform the DNA quantification. However, it is a library we have previously used and was measured recently by 3 separate people, all who came up with the same concentration.

[pmiguel]

[...]

I did not perform the DNA quantification. However, it is a library we have previously used and was measured recently by 3 separate people, all who came up with the same concentration.

Were they all using the same assay? What was the method?

--
Phillip

[suefo]

I don't think they used the same method. I believe the CORE facility who did the original FLX run used qPCR. One of the other methods may (and I need to confirm this) used a pico-green kit?

[suefo]

I don't think they used the same method. I believe the CORE facility who did the original FLX run used qPCR. One of the other methods may (and I need to confirm this) used a pico-green kit?

Actually I just learned that we use the Agilent to obtain concentration as well as purity.

[pmiguel]

Could you post the electropherograms?

You mention "amplicon" in your first post. Do you mean you used PCR to produce the library you add to your emPCR?

Also, sometimes during PCR small primer dimer/adapter dimer amplicons become complexed with the larger amplicons. So, even after size selection, they persist in your library. You can assay for this by running some of your library denatured on a RNA Bioanalyzer chip. (Denature at 95 oC then put directly on ice until you are ready to load the chip.)

--
Phillip

[mohdsos]

I would recommend double ampure beads selection. or gel purification,

the dimer can cause your enrichment to be high. once I had more that 80% enrichment.


Mo

[suefo]

@Phillip... yes we first generate PCR products. These samples are analyzed individual first on an e GEL to confirm DNA presence and then on the Agilent post-AMPure step to exam size, concentration and purity. Samples are then pulled using the same number of molecules (96 separate samples in all), then re-purified using AMPure and then another high-sensitivity Agilent looking for any primer dimers that may remain before continuing with emPCR. So you would suggest that the Agilent is unable to "see" the dimer due to being complexed with our fragments - which appear to be the correct size (or size expected)?

@Mo... we will look at both your's and Phillip's suggestions to see if there is this "hidden" dimer that is complexing with our amplicons.

[pmiguel]

Yep, that is what I think can happen. Assaying for that is problematic however. The problem being, well for me at least, I expect a more or less agarose/EtBr/TBE-like result from electrophoresis. But Agilient chips give this bizarro/non-euclidean electrophoresis results where a double-stranded fragment will run faster than a single-stranded fragment. See earlier threads:

http://seqanswers.com/forums/showthread.php?t=12852

http://seqanswers.com/forums/showthread.php?t=12523

for evidence of this. As a result just running a pico RNA chip on a denatured sample doesn't really make everything clear. I mean, as always, some of the fragments are not amplicons. But also, some of the denatured amplicons may renature just prior to loading on the pico RNA chip, or even during electrophoresis. Or, I fear they may. So if I see fragments below the desired size of the library is that real, or a mirage?

Here, for example, is a specialized library made by another lab run on a HS DNA bioanalyzer chip:



Looks pretty good, right? Now look at the same library, denatured and run on a pico RNA chip:



(I zoomed in a little, so note the scale at the bottom.) Are the 74 and 120 nt fragments amplicons? Maybe even the 46 nt one? Hard to say. But, I think they are. Here is the SAV % called base from the 100 base read:



Amplicon! Probably a primer dimer. But there was something there on the HS DNA chip.

So how does one handle this? Not sure yet.

--
Phillip

[relaswar]

Could you post the electropherograms?

You mention "amplicon" in your first post. Do you mean you used PCR to produce the library you add to your emPCR?

Also, sometimes during PCR small primer dimer/adapter dimer amplicons become complexed with the larger amplicons. So, even after size selection, they persist in your library. You can assay for this by running some of your library denatured on a RNA Bioanalyzer chip. (Denature at 95 oC then put directly on ice until you are ready to load the chip.)

--
Phillip

Which RNA chip do you recommend? I am having similar problems with primer dimers.

[proteasome]

I've had issues using the bioanalyzer to both quantify and qualify my products. I'd recommend a picogreen-based quantification method instead of the chip.

[MissDNA]

Before Roche introduced RL protocol with double stranded libraries, we always based our library quantification on the RNA Pico chip results and things seemed to work better. No crazy cpb/enrichment. We still quantify our PE libraries on RNA chips, and things work fine.
However for the new libraries that are double strand we use HS chips to qualify and Roche´s recommended fluorometer to quantify.

[pmiguel]

I've had issues using the bioanalyzer to both quantify and qualify my products. I'd recommend a picogreen-based quantification method instead of the chip.

picogreen basically gives you a count of the number of nucleotides in double stranded molecules. If, for example, 10% of those nucleotides compose short amplicons (1/10th the length of the long amplicons -- primer dimers) then your estimation of "copies" of amplicons will be a factor of 2 low. Same issue for SYBR green based qPCR.

--
Phillip

[pmiguel]

Which RNA chip do you recommend? I am having similar problems with primer dimers.

Agilent RNA pico chip. Problem is that the sample will reanneal somewhat prior to/during loading. So you can get complex shapes for your full length products. These do not necessarily reflect the true lengths of your products. (For rRNA ITS work, I have seen two completely different sized peaks after denaturation. Neither of which seems to be the right size.)

Anyway, not the point of running this type of chip: you are looking for small "hitch-hiking" primer dimers hiding amongst your full length amplicons by anneal to their adapter ends.

Alas, the RNA chip analysis software SUCKS compared to the DNA chip software for estimating peak molarities. So you are stuck doing that by hand.

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Phillip