Viruses, Vol. 16, Pages 636: U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing

Viruses, Vol. 16, Pages 636: U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing Viruses doi: 10.3390/v16040636 Authors: Jennifer Diaz John Sears Che-Kang Chang Jane Burdick Isabella Law Wes Sanders Colton Linnertz Paul Sylvester Nathaniel Moorman Martin T. Ferris Mark T. Heise RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5′ conserved sequence element that disrupt sequence but not structure did not affect ...
Source: Viruses - Category: Virology Authors: Tags: Article Source Type: research