Table 1. Experimental design–challenges and solutions.

Challenge	Solution
1. Replication-initiating viruses might contain variable numbers of founding errors	Initiate replication with RNA transcribed by host Pol II that incurs one round of errors at a constant rate.
2. Viral genomes might have replicated in a variable number of cells prior to sampling	Restrict replication in single cells by using a plant-infecting virus (TCV) with MP genes disrupted.
3. Phenotype-based selection might enrich certain mutations	Profile errors in the non-protein-coding (-)-strand replication intermediates.
4. Differentiate between primary and secondary (-) strands	Generate and analyze continuous cDNA fragments of at least 2,000 nt in length.
5. Ensure cDNA of (-) strands is exclusively derived from (-) strands	a. Carry out strand-specific RT-PCR following an established procedure; b. Include control constructs that produce exclusively (+) and (-) strands, respectively.
6. Account for errors incurred during all steps of the error-profiling experiment	Include appropriate controls at every step. Especially relevant to current study was the RTRC control producing (-) strands independent of viral replication.