Fig. 1: Creation of molecularly barcoded influenza A virus populations.
a Molecular barcodes containing 10 randomized nucleotides were encoded downstream of the open reading frame in the PA and HA genes, shown as cRNAs. A registration mark was also added to HA to distinguish unique barcode libraries. Sequences were repeated downstream of the barcode to maintain contiguous packaging signals required for replication, and silent mutations were introduced into the open reading frame to avoid direct repeats. b Experimental overview where randomized barcodes were cloned into reverse genetics vectors followed by large-scale, parallel virus rescues to ensure unbiased barcode distribution. c Optimized rescue plasmids enhance viral yield. Rescue efficiency was determined by measuring viral titers at the indicated times post-transfection with the standard 8-plasmid system, a consolidated 3-plasmid system, or the 3-plasmid system plus a vector expressing TMPRSS2. (data presented as the mean of n = 3 ± sd. ANOVA with Tukey’s post hoc, *p < 0.05 relative to 8-plasmid rescue, #p < 0.05 relative to three-plasmid system.) Source data are provided as a Source Data file.
