Hi Toby,

I've used the older Q24 quite a bit for methylation assays, but not the Q48. We've found that gene-specific assays can be variable and sometimes require going off book from the manual.

Low peak height can be due to several variable. Here are some steps that we have used to improve low peak height.

1. Take a look at your PCR products on a gel to be sure that you're getting a good, robust product. Optimizing PCR, specifically annealing temp, solves ~75% of our low peak problems.

2. Since the bisulfite converted DNA is extremely low quality template for PCR, sometime it is not possible to get robust bands with your primers. If your PCR bands are wispy, you can definitely use more than 10 uL. We sometime use up to 20 uL for our reactions. For 1 particular assay, we run duplicate PCRs and combine them for 30 uL reactions.

3. If you're getting good PCR product but still have low peaks, consider designing a set of alternative sequencing primers. It may be that the seq primer is not optimally annealing to your PCR products.

I hope this helps.

-Ray

Ray A Enke . Do you mean you use more than 10ul in the Q48 disk?

I do have the same problem,

Did you find a solution ?

Thanks a lot

No solution

ran all samples on the q24 in the end at a different lab.

common problem i think…

I am also trying to troubleshoot this issue currently. We are moving an assay that had previously been optimized for Q24, and is now struggling with low peak height on Q48. I have found that increasing the concentration of template in the initial PCR (doubling uL of DNA extract), and adding more cycles to the program, mostly resolves peak height issues (we doubled peak height). This will increase DNA concentration.

If you just increase uL PCR product into pyrosequencing disc you will dilute the beads and other sequencing reagents, which will also reduce peak heights.

Our peak height is improved, but often inconsistent due to another issue, I think, and I'm curious if anyone else has had this issue as well. The enzyme well on three brand new cartridges is constantly "clogged" such that I wonder if it is really clogged, and maybe an electrical signaling issue. We also have regular errors about dispensations of E during pyrosequencing, which I think causes some peaks to be lower than expected, messing up methylation analysis. Does anyone else have this issue?

Toby Candler Sarah Dean Carole Grenier Amelie Taschereau

Hi all,

I recently purchased a Q48 and have also been frustrated that at the 10 µL volume limit on the disk. I've found that I can get away with loading up to 12 µL, but that often doesn't solve the problem for amplicons that have low peak hight. Two workarounds that should work:

1. Use a speed vac set up to evaporate 20-30 µL of PCR amplicon down to ~10 µL

2. Use a silica column PCR cleanup kit to concentrate 20-30 µL of PCR amplicon down to 10 µL

I wish my Q24 had never broken!

Hi, we're using the Pyromark Q48 for methylation too, and also for SNP genotyping, but we're having a different type of problem with it. After the sample run is successfully completed, the spaces dedicated to the enzyme, to the substrate, or to the denaturation solution alternately are not emptied from the cartridge. We tried to do several washing to unlock the cartridge but the problem came back again. Does anyone have the same problem? Thank you

I also had this issue but now after the PCR step, I use a speedvac to concentrate samples further and have good results. I also use 10uM of sequencing primer instead of the suggested 4uM.

Elisabetta D'Aversa have you tried cleaning the cartridge with 70% or 80% ethanol for 30 mins before a run? I do this sometimes, then do three wash runs (with water) and the cartridge is good to go and doesn't clog during the actual run.