Excess PCR primers in final product

[PaulineF]

Hi,

Has anyone ever had primers that were too concentrated for the paired-end sample prep? We are getting excess primers left over after the PCR, which is after the gel purification. But the excess primers were not there before the PCR. Moreover, our first batch of PE sample preps were fine (i.e. no excess primers).

PF

[ECO]

I don't understand your question. Sounds like you got primer-dimer or non-specific products, rather than primers that were too concentrated.

[PaulineF]

The whole story was the new PE protocol says to run 10 cycles of PCR and when I first did that, all I got was an amplification of primers because the cycles were not long enough to amplify the actual sample. Then I increased the cycle number to 16 and saw my sample smear between 200-350 bp but in addition, there was a band around 77bp that is consistent across all samples corresponding to the primer peak. The point is we have run similar samples through another PE kit before and never got the extra primer peak. I ran the remainder of that old primer with the new primer on the Bioanalyzer and found the new primer to be aobut 5x more concentrated, but the readings between the various tubes of new primer varied a little.

My question is if anyone has had experience with primers that may seem too concentrated, resulting in excess amounts in the PCR product. Do you know what the stock concentrations of PE primers are?

[suludana]

Hi,
the same thing happened to me. And then, when I cleaned the PCR reaction (Qiagen 28104) I lose the entire smear of 100-200 bp, leaving only some of the band corresponding to the primers. What should I do?

[PaulineF]

Hi Suludana,

May I ask what are the lot numbers of your primers in question? When is the expiration date of your kit? Just curious. I had my lots checked by Illumina's R&D and they claimed those lots were fine. I tried decreasing the primer concentration going into my 18-cycle PCR reaction but only some samples had product, and for those, there was no more primer peak on the Bioanalyzer.

Pauline

[suludana]

Well, actually I was making a handmade sample preparation for indexing. I was not using an Illumina kit, which does not yet exist for indexing. But Illumina gave me the concentration of primers (25 uM) and I was working with it.

I find it very strange that the primers are positioned at about 100 bp and the higher band get away when I clean the PCR.

[PaulineF]

I was getting what is most likely primer dimers at around 77bp, that's probably what you have too. But I do standardly put my PCR product through the Qiagen kit to purify it (as per the Illumina protocol) and I don't lose my sample that way.

[suludana]

Hi,
why do you talk about primer dimers of 77 bp? How can you explain this size? One of the primers have 58 bp and the other 34 bp. And they are not complementary.

[PaulineF]

Hi,

I've been working with the Illumina kits and I am not indexing. I do not have the exact size of the primers but primer dimer was also suggested by Illumina to be what my smaller bands may be as they claim the primers are over 30nt long. I am sure though that primers for multiplexing are different from single sample PE primers. So I am not sure if you are having the same problem that I am.

[suludana]

It is true that I am doing indexed, but I'm using primers of the standard protocol (58 and 34 bp). The difference is only in 1 nucleotide (I do not have tags on my PCR primers) ... so the size is almost the same ... I see my primers in a position of 100 bp and I don't understand it ...

[cjohns]

I have done several chip-seq with illumina kits and on my last set I also got a product that is about 80bp long that I have not seen before. It is in my blank gel that I amplified too, so it has to be from the amplification primers. My primers from different kits seem to have similar concentrations according to the spec I used. It is very odd that all of a sudden I am getting primer dimers now. Has anyone figured out how to fix this problem?

[PaulineF]

I heard that somehow in the gel purification step, if you use the Qiagen kit as Illumina suggested, instead of heating your gel+buffer mix at 50C, vortex at high speed for 5 mins at RT and that somehow helps prevent concatemers. I have yet to try it out, but I heard it works.

But yes, I suddenly started to get these small peaks too, samples are similar enough to what I've run before and we're still supposedly buying the same kits. Weird...

[mjg54]

I completed my first sample prep and I see a band just below 100bp, I think it is an amplicon product from the primers priming one another. I was wondering if this will be sequenced, because it is so short it may not undergo bridge amplification.

[cjohns]

No, they do not get sequenced. Our only concern is we use PicoGreen to quantitate and it would detect these amplicon products and we may not have reliable quantitation of our insert DNA.

[mjg54]

so, what is the 100bp band?
I can estimate from a gel what percentage of my sample is the "sequencable" smear and what is the 100 bp band, then when I quantify I can normalize.

thanks for the extremely quick response

[BioHak]

If you believe it is an amp by-product, why not sequence a sample of it on a capillary instrument and verify?

If your goal is to remove the smaller fragment, we cleanup our samples with the Aggencourt AmPure product? We see significant dropoff of any products under ~90bps and nearly 100% recovery.

[captainentropy]

Unless there is something going on that I haven't figured out yet I think the peaks you are describing for the PE amplification are amplified primers as I am seeing them too. However, the size of the peaks are larger, around 125 bp, though there is a tiny peak around 66 bp as well. In previous libraries I've seen a very strong 150-160bp peak that I know was amplified adapters that resulted from me cutting too far down on the gel after the ligation step. I've already sent these current samples for sequencing, I'll know for sure in a week or two whether they are adapters or primers.

In the attached images are my last two libraries. One is a library made from 100ng of input DNA and the other I used 1000 ng. Other than that and the day I made them on they are identical in construction (also, I use a 1:30 dilution of adapter instead of 1:10). Another person in my lab is using the same paired-end protocol and he has seen the same pattern. It was also present in a no-template control reaction, so we're confident it's just amplified primers. He has been optimizing the PCR step and has found that raising the annealing temp didn't prevent the peak from appearing and neither did adding DMSO to the reaction. The only thing that worked for him was to optimize the primer concentration. The concentration of the primers Illumina provides (or tells you to use if you ask them) is 25 uM. Thus in the PCR the [final] = 1 uM. For standard PCR I've always used [final] = 0.4 uM, and that is for 25 or more cycles! Although I am using the PE adapters/primers I've been using 18 cycles of PCR as written for the single-end adapter protocol. I'm not sure if I used 12 cycles as written for the PE protocol that it would make much difference as the products amplified in the first few cycles are most important for the final outcome. I can always just run the PCR on a gel and purify the library from the primers but the problem is at the PCR step. I think the primer concentration Illumina is suggesting is too high. Any opinions on that?

[csoong]

The pre-PCR gel extracted product should be characterized in terms of OD ratio and concentration and this would act as a validation step prior to PCR. This pre-PCR quality may greatly influence the required amount of starting materials and number of thermal cycles required for PCR-ing. If we could share info about pre-PCR characterization, post PCR characterization, insert size, and PCR settings on successful sequence runs, this would be very helpful to the community.

[captainentropy]

agreed csoong. However, if you are using ChiPed DNA you might only have a few nanograms to work with. That is hard too quantitate much less get a 260/280 ratio.

In my lab we're pretty sure the peaks we are/were seeing come from the PCR primers. Once I get my sequence back I should know for sure though.

[JUdw]

Originally Posted by captainentropy

....The concentration of the primers Illumina provides (or tells you to use if you ask them) is 25 uM. Thus in the PCR the [final] = 1 uM. For standard PCR I've always used [final] = 0.4 uM, and that is for 25 or more cycles!

1ul [25uM] in 50ul PCR reaction, [final] = 0.5uM