Pyrosequencing is a method of sequencing that relies on light emitted in an enzymatic reaction set in motion by the release of pyrophosphate when a base is added during the sequencing process. The results are quantitative, making the method ideal for sequencing samples of known sequence that may have mutations in proportions as low as 5%, such as tumor samples or mixed viral populations. Test kits exist for APOE, BRAF, KRAS, HFE and MTHFR, but any small region (around 80 bases) can be analyzed with custom primers.
This method can also be used for characterization of contiguous and multivariable mutations, LOH analysis, unambiguous, fully quantitative genotyping that distinguished multi-site variations from single nucleotide polymorphisms.
Microbial identification and drug resistance typing are also potential uses of this method. The system provides rapid and reliable high-throughput screening. Mutations can be accurately identified without use of expensive labels and dyes.
Specific regions of methylation can be assayed via sequencing on the Pyromark Q24 system as well. Bisulfite converted DNA is PCR amplified with a biotinylated primer pair. The non-biotinylated strand is sequenced in the Pyromark system. Custom regions can be amplified, with specific analysis software is available for use by customers. Qiagen also has over 30,000 assays designed for CpG Islands throughout the human genome, which can be ordered through our Geneglobe.com service.
Assay design and analysis software is available for use by any Johns Hopkins investigator. Software must be run on a Windows XP or higher machine with a GB of RAM available.
Investigators provide PCR products and sequencing primers to the lab for sequencing. We can provide PCR support services for an additional charge. Qiagen kits can be ordered at a slight discount and free shipping by e-mailing your GeneGlobe.com shopping cart to firstname.lastname@example.org.
To place an order, use the Pyrosequencing order form on the JHU ordering server. You will need to upload a picture of your quantitation gel for the samples. For new custom assays you will also need to upload the .xml file generated by the pyrosequencing primer design software or to fill out and provide the appropriate assay information form from the Downloads tab on the left on this page. Please bring 20-40 ul of sample (depending on how bright the band is on a gel) and the lyophilized primer from Qiagen. If you ordered your primer from someone other then Qiagen or don’t wish to bring over the entire sequencing primer, then please contact us for the appropriate resuspension buffer. Primers should not be resuspended in water.
Samples must be dropped off before 4:30. Results will be available on the JHU ordering server within 48 hours.
- A .tsv (tab-delimited) file which lists each query site and the percentage of the bases queried
- A .pdf file which shows the peaks generated and helps to explain error messages and other information about the run.