How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures that are extended or the primers itself form dimmers that serve as template. Perform accurate sequencing controls (e.g., PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and, if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.