Fig 1. Experimental setup for HAdv5 mutation rate measurement.

Three serial endpoint dilution transfers were carried out in 96-well plates (hypothetical infected wells are depicted in red) and then passaged twice in 10-cm plates at high MOI. Each endpoint dilution should remove pre-existing diversity. The estimated number of infection cycles during which mutations could accumulate are shown below: two for growth of the virus from a single PFU in the last endpoint dilution step, and one for each high-MOI transfer. Viral DNA was purified and used directly for Duplex Sequencing. The entire experiment (endpoint dilutions, amplification, and sequencing) was repeated three times (R1, R2, and R3).