How are difficult sequences/regions or failed PCR handled?
• Computationally identified problematic regions in the initial reference sequence such as repeat motifs and high-GC content sequences. These regions are masked to exclude these sequences in the design of PCR primers; • Designing multiple primer pairs to target difficult regions; • Attempting alternative PCR chemistries and thermocycling parameters for the failed region; and • If a region fails two attempts with alternative conditions, that region is considered refractory to analysis. In general, 10 to 15 percent of targeted regions are refractory in this manner.
