Which kit do you use for pyrosequencing? I have performed experiments with Pyromark (Qiagen), and the problem that I had many times was the enzymes that I used. The enzymes that are used, are very sensitive. Don't you have any positive control for check the enzymes?

just have a look on the invoice from your primer supplier. They should contain the sequences and the modifications. The beads have a size of 34 µm, they will not move in your gel.

Do you get the first Peak when the Enzyme and Substrate have been injected, so right before the first nucleotide is injected? If not, the problem might lay here, maybe due to a needle (from the cartidge) that is clogged...

Thank you for your replies!

I am using Pyromark and I have run another assay in between runs which has been successful so it appears that the enzyme and substrate are fine. I also test the tip dispensation before each run. The primer set has worked previously so I am wondering if the primers are faulty (we re-ordered the same primers which were fine before) as a batch issue? Does anyone know if there is any way to test if the primers are biontinylated as stated?

I have had similar problems with assays that worked previously and it has mostly been due to the substrate/enzyme not being dispensed at the beginning of the run. Try another cartridge or clean the cartridge with warm water (high pure) and leave at 37 degrees for about 1-2hrs before loading the nucleotides, enzyme and substrate. Also, once loaded tap on the bench, gently, a few times before loading the cartridge into the machine. Do you allow the primer-PCR product mix enough time to cool down prior to running?

Sounds like a primer issue, I would contact the company that supplied them and let them know they aren't working correctly. Do you have any results generated using the old primers that you could sent to them to show that the assay has worked before? Are you amplifying your target using DNA isolated from similar cell/tissue types as before?

Thank you all for your input!

Plates usually get about 10-20mins to cool down before they are run.

I am using the same source of DNA as before and the same samples work fine on other pyrosequencing assays.

It is looking more and more likely the cause is due to the primers and perhaps the best thing to do would be to attempt the assay with new primers and hope that solves the issue! I just wanted to make sure there wasn't an obvious area I wasn't considering or had missed something!

I am 99% sure is the biotinilated primer that does not work.. especially if the assay was working previously.. I had that once (by mistake a I order my biotinilated primer without biotinilation..)

I suggest to use primers from IDT (Integrated DNA technologies) they are great..and never make mistakes.. and if you don't want to change company you definitively need to order new primers. Good luck!

Ah! I forgot... there is no need of HPLC purification... if you buy them from a company that only produce primers you can be sure that they are of great quality...

HPLC purification it's something invented by some companies to make money so that instead of paying 25 dollars for a set of primers you pay 150.

We have in the lab primers HLPC purified and they are much worse than the "normal" ones.

Sorry for the extra comment.ciao!

Hey Kristina

As I understand your enzyme , substrate and dNTP's work well .I am beginning to think that you dont have enough starting material after the PCR. You can very well run the samples on the gel and observe the image i have done that and biotin has not impacted the gel by any means. I agree with Benedetta Izzi IDT is a good choice for pyrosequencing primers.

One suggestion you should try and replace ethidium bromide with gel red far more safer option for obvious reasons.

Hope this helped

I think Alan O'Doherty has a good point. If you have got PCR bands on gel, then the problem probably lies on the post-PCR reactions. In my experience (13 years with Pyrosequencing) the reaction cartridge is often a source of errors. I would recommend you to replace the cartridge or carry cleaning steps as those recommended by Alan O'Doherty.

Another common source of errors in pyrosequencing is the probes used to collect the beads during the ssDNA preps and washing steps. You may perhaps try to install new probes. But first and foremost, make sure that you have PCR bands on gel. Why not run a control DNA sample that worked before to make sure that your samples are OK?

Good luck!

Is it possible that the expected sequence of bases has been altered or input incorrectly into the Pyromark software? If the software is expecting bases that aren't in the right sequence, this may explain the lack of peaks.

Firstly I would check that the primers ordered are correct e.g. sequence, biotin modification etc... We have done this before, particularly when we have one PCR product, but multiple pyrosequencing primers to sequence the entire product - we have used the biotin on the wrong PCR primer - I've also seen copy / paste errors where F/R primers were ordered with the same sequence.......... Then I would check that the assay is correct on the machine and that you do not have any errors in the dispensation order.

At this point, I would then look at the recent results that didn't work. Do you have the initial Enzyme / substrate peak? - if not, clean the cartridge and try the run again with new E / S if necessary, and perhaps a new cartridge.

If you have the E/S peak, the primers look OK, and the assay is exactly as previously used, then I would have one of your PCR products Sanger sequenced (if you have good access to a Sanger sequencer......). My experience is that primer manufacturers make exactly what you ask them to make. Over the last 20 years I don't think that I have ever had an incorrect primer delivered - but I have had many PCR products of the correct size, that were not the anticipated product / sequence.

HTH

Jon

So I ordered new forward and reverse PCR primers and my assay is working again. All the correct sequences were sent for the previous order so either there was a fluke error with the batch of primers or there was some sort of labelling mix up upon arrival into the lab. Either way, the new primers are fine and I know a lot more about possible pyrosequencing errors for future analysis! And at Viva defense...

Thank you for all your contributions and help!

Kristina.

Dear Kristina and collegues,

normally I do not like to reactivate old discussions (2013 !) but this topic fits exactly the problem we have in our lab with one single pyrosequencing assay atm.

The assay worked well in 2014, but we are not able to get comparable results yet.

Same Primer, same buffer, same machine. We reordered the primer two times (same company) but the results are again of bad quality and weak signals.

The PCR bands are better compared to 2014.

Other assays work well (same cartridge).

The positive control (we use the commercially available positive control "primer" from supplier Q) gives comparable good signal quality like in 2014.

We have no clue what to change.

I wonder if there might be a problem at the level of the biotin / sepharose-beads?

Maybe there is a problem in binding of the biotinylated PCR product to the beads?

I found this link in the internet:

https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/09/20/which-biotin-modification-to-use-

Does anybody have some experience with modifications of the biotin in pyrosequencing or other suggestions like different kinds of sepharose beads?

Thank you all for some input in advance.

Axel

From the IDT webpage you link to, you want the standard 5' biotin, nothing fancy.

HTH

Jon

Yes, that is what we order in general.

Do you have experience with some kinds of modifications of the biotin molecule?

Do you have experience with different kinds of sepharose beads?

We did some experiments in between.

We repeated the PCR with bisulfite treated DNA from samples that worked quite well and samples that showed really bad signal quality in the pyrosequencing in the runs before. The PCR bands in the agarose gel look all the same!

In the pyrosequencing the signal quality is reproducable in all cases. Bad in samples with former bad quality and good in samples with former good quality.

I have really no clue which parameter/step might be the responsible one.