Step	Cycle	Temperature	Time
Initial denaturation	1	98°C	2 min
Denaturation	25–30	98°C	30 s
Annealing ∗		55°C	30 s
Extension		72°C	30 s
Final extension	1	72°C	2 min

∗Annealing temperature indicated is optimized based on the Tm of the SPseqF4 and IRESrev primers. Typically, annealing temperature is Tm – 5°C.