Universal Competition Assay by Nanopore Sequencing (U-CAN-Seq) v1

Competition assays are an effective and rigorous method that can be used to understand the relative fitness of different viral genotypes. In competition assays, two genotypes of virus are used to infect the same cell culture well or animal, replication occurs, and the relative abundance of each genotype is measured to determine the relative fitness of the two viral genotypes. However, it is technically challenging to distinguish between and quantify very similar genotypes of a virus. Here, we describe a protocol combining RT-PCR with newly available commercial nanopore sequencing services to perform competition assays on RNA viruses. Our assay, Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively inexpensive and sensitively detects fitness differences between two similar genotypes of a virus. Competition assays using the U-CAN-seq approach can be divided into four parts: infection, RNA extraction, RT-PCR, and sequencing and analysis. First, we infect cells at a multiplicity of infection (MOI) of 0.01 using each virus genotype individually and different ratios of the two genotypes. We sample the inocula before infection and the cell supernatants at 24 hours post infection (hpi), extract RNA using Trizol-LS, and then use gene-specific primers to generate cDNA. We use PCR to amplify the region of the viral genome that differs between the two genotypes, and we submit the linear amplicons for commercial nanopore sequencing. Finally, we developed code to provide a simplified method to search for strings of sequence unique to each genotype and calculate the ratio of the genotype of interest to the total read counts at that locus.