Genotype by Barcode Sequencing of 10 experimental coinfections among 5 influenza A virus strains
We conducted infections following the conditions of Marshall et al. (2013). Briefly, a 6-well plate with 1 X 106 of MDCK cells was coinfected with a pair of strains in equal proportion at a MOI of 10. We synchronized infections at 4C for 1 hour to promote equal attachment of viruses. To limit infections beyond one cycle of replication, we conducted infections in the absence of trypsin (Klenk et al., 1975; Tobita et al., 1975) collected viral supernatants at 12hr post infection and stored them at 80C for subsequent genotyping and titration.Control InfectionsWe conducted several control experiments to test the genotype by barcode sequencing approach (see below). First, we made biological replicates of the same coinfections for three strain pairs (HK68xCA09 n = 2, and HK68xPAN99 n = 2, and CA09/PAN99 n=3). Second, we conducted infections of HK68xCA09 at increasing MOIs (0.01, 1, and 10), which are expected to lead to increasing levels of coinfection and reassortment (Marshall et al. 2013). Finally, we evaluated the parental stocks used to initiate coinfections to evaluate the genotyping pipeline and establish thresholds for assigning sequencing reads to specific strains.To isolate clonal populations for genotyping progeny from the infections, we conducted plaque assays. Plaques were picked with 1mL sterile pipette tips and placed in DPBS in deep-well 96-position plates for genotyping (see below). We also conducted plaque assays from the same supernatants to quantify infectious particle production yield of experimental coinfection supernatants.To ascertain the eight-segment genotype of the viral progeny (i.e. isolated plaques representing clonal populations), we used a strategy we term Genotype by Barcode Sequencing (GbBSeq), inspired by Bar-seq experiments of yeast mutant libraries (Robinson et al. 2014). We describe the strategy in detail in Supplementary Material. Briefly, the picked plaques are used as RNA template in a one-step RT-PCR reaction with: 1) a uni13 primer (targeting a conserved region shared by all influenza A virus segment ends) barcoded to correspond with each of 96 wells; and 2) 10 primers (6 for internal segments and 4 primers customized for H1/H3 and N1/N2 subtypes), targeting the internal regions of genome segments barcoded to correspond with each coinfection (represented by a plate). These barcoded PCR products were pooled, diluted, and used as template in a second PCR reaction that added adapters for paired-end sequencing on the Illumina MiSeq platform. All GbBSeq experiments in this paper (including controls) were conducted in a single MiSeq run.