Influenza mutation rates using PrimerID method

We were interested in testing if Illumina sequencing could be reliably used to measure the mutation rate of influenza virus. To do this we used the Primer ID method to sequence large number of individual viral genomes and assessed the frequency of nonsense mutations in the PA gene within the population. We first needed to verify the signal to noise of the assay and that the reverse transcription step of sample preparation did not introduce too many errors. We set up 2 samples to test this. The first was RNA from the supernatants of virally infected cells. Mutations observed in this sample would be from the viral RdRp and the RT step. the second sample was from cells transfected with a plasmid expressing the RNA of an influenza gene. Mutations observed in this sample would be from RNA polI and the RT step. Comparing the mutation rates of the two samples will inform us of how much background mutational signal RT contributes.