Targeted Amplicon Sequencing and Data Analysis

MDCK cells were pre-treated with 0.25 μM SMeFM or 0.50 μM T-705 for 24 h. The pretreated cells were infected with the influenza A virus (PR8) at an MOI of 0.1 PFU/cell for 1 h and incubated at 37°C with 0.25 μM SMeFM or 0.50 μM T-705. Total RNA was extracted using ISOGEN reagent (Nippon Gene, Tokyo, Japan) at 8 hpi, and cDNA was synthesized with the Uni12 primer using ReverTra Ace (Toyobo, Osaka, Japan). The fragment of segment 7 vRNA (nucleotide position 406–640) was amplified with specific primers using KOD One (Toyobo). A second PCR was performed with adaptor and index primers using KOD One (Toyobo). Sequencing was performed using a MiSeq sequencer (Illumina San Diego, CA, United States) with MiSeq Reagent Micro Kit v2 (300 cycles) (illumine). Read sequences were filtered according to base quality using Trimmomatic (q = 30) (Bolger et al., 2014). The filtered sequences were aligned, and the ratio of nucleotides in each position was calculated. A chi-square test was used to determine the statistical significance of mutation rate between DMSO treated cells and compound treated cells.