7 ways to optimize your pyrosequences (PyroMark, Qiagen)

When talking or reading about pyrosequencing you're probably thinking immediately of the high-throughput 454 sequencing. This sequencing technique is based on the "sequencing by synthesis" principle and differs radically from the standard Sanger sequencing. It can be viewed as the qPCR version of standard PCR. This technique was commercialized by Pyrosequencing AB and has been further developed for high throughput sequencing by 454 Life Sciences. Qiagen acquired the pyrosequencing business line in 2008 and started developing the pyrosequence technique for low/medium throughput applications.  So, even though it is based on the same technology (see video below), the pyrosequencing I am talking about here is NOT the 454 system but the Qiagen system!

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In the video above the pyrosequencing reaction is shown in an animated way with the graph output as you would get with Qiagen pyrosequencing software.

I have used pyrosquencing to determine the ratio of two Single Nucleotide Polymorphisms (SNPs) at a certain genomic position. In my case I was dealing with a ratio of C/T SNPs in a 100 bp genomic region  after bisulfite conversion of my genomic DNA. The technique is quite simple but also prone to errors if you don't take into account a number of optimisation steps. In this post I assume that you know the Qiagen pyrosequencing technique but you've run into some issues during the assay testing phase as I have.

Here, I give six ideas on how to optimize or troubleshoot your pyrosequencing assays!

1. So, you have designed your assays and now you want to order the primers. Biotinylated primers are very expensive and you don't know if your primers will amplify your target region without by-products. Therefore, first of all, you will start testing your pyro-assays with normal non-labeled primers.  But, choose your primer manufacturing company wisely! Cheap manufactures produce low quality primers and in many cases you get less primers than what you asked for and -worse- then you think you have, so your primer dilutions will not be accurate. All this can lead to spurious PCR amplification which is visible on an agarose gel as extra fragments or smears. You can buy primers for a custom assay at Qiagen but they will only deliver the primer-set including the biotinylated primer.
   
   
2. Now, you have good primers but you still have extra by-products in your PCR. Did you already run a temperature gradient PCR? Maybe try again but now with higher annealing temperatures in a range of 58C to 65C, you would not expect a template with additional uracil/thymines from the bisulfite conversion to have this issue but my assays improved markedly when I ran them at 61C instead of the standard 58C.
   
   
3. Still suffering from primer dimers? Maybe your template DNA is low in quality and/or concentration and the primers are not used up in your PCR reaction. Try lowering the amount of primer in the reaction in steps of 50nM. In my case, I had to use only 75nM of each primer to reduce the amount of primer dimers in the reaction.
   
   
4. Keep in mind that your bisulfite converted DNA is single stranded and way more sensitive to degradation. Best practise is to aliquot your DNA into PCR plates and store them at -20C. For each PCR you can then just take one PCR plate with your DNA from the freezer and use it. Make sure though that you seal your plates really good with a proper adhesive to prevent freeze drying your DNA.
   
   
5. Now, lets assume that you have tested your primers and that you have a few assays that produce nice bright single bands. The next step is to retest your protocol with biotinylated primers. Make sure that you order these primers again with a respectable company and that you HPLC purify these primers. That's the only way to make sure that the the majority of the primers indeed have the Biotin label and that there is no free Biotin in your primer. Biotinylated primers are also really sensitive to repeated freeze-thaw cycles (max. 3 times) so aliquot these as well.
   
   
6. If you tried everything to get a good PCR amplification and nothing works? Redesign your assay! Sometimes primers will just not work for a variety of reasons such as secondary structures in your DNA and you can spend ages getting it to work while a new primer set will just give a good amplification from the moment you use them. Also, when in doubt, change all your reagents and use new stuff. You never know what happened with a certain tube (too long on your bench, colleagues messing with it, etc.).
   
   
7. The last tip but a very important one: check your pyrosequence target regions for insertions/deletions and other variations such a SNPs. You have to make sure that the sequence you provide the PyroMark software is the correct one without any possible variation other than the variation you give it. You may have to first Sanger sequence the all your sample fragments to be absolutely sure, depending if you work with highly inbred samples or with samples from the field that are highly variable. If during sequencing a nucleotide position is found that doesn't match your dispensation order the whole run will be messed up!
   
   

Hopefully these tips can be of use when your doing your pyrosequencing. Let me know! Or if you have additions to these tips from your struggles in the lab leave a comment!