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Updated: Jun 27, 2025

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Published on: March 11, 2020

U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing.

Jennifer Diaz¹, John Sears¹, Che-Kang Chang¹

¹Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA.

|April 27, 2024

Summary

This summary is machine-generated.

A new Universal Competition Assay by Nanopore Sequencing (U-CAN-seq) quickly and cheaply measures RNA virus fitness. This method reveals how viral mutations impact fitness, aiding in understanding virus evolution and human health risks.

Keywords:

RNA virus alphavirus competition assay nanopore sequencing viral fitness

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Last Updated: Jun 27, 2025

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Published on: March 11, 2020

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Published on: June 3, 2019

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Published on: March 15, 2019

Area of Science:

Virology
Evolutionary Biology
Molecular Biology

Background:

RNA viruses rapidly evolve, impacting viral fitness and host range, posing risks to human health.
Assessing the evolutionary fitness of novel viral variants is crucial for understanding epidemic potential.
Traditional competition assays face challenges in quickly and affordably quantifying fitness differences between similar viral genotypes.

Purpose of the Study:

To develop a cost-effective and sensitive method for assessing RNA virus evolutionary fitness.
To enable rapid quantification of fitness differences between closely related viral genotypes.
To investigate the impact of specific mutations on chikungunya virus (CHIKV) fitness.

Main Methods:

Development of the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq).
Utilizing reverse transcription PCR and nanopore sequencing for competition assays on untagged RNA viruses.
Application of U-CAN-seq to study a known CHIKV mutation (N24A) and mutations in the 5' conserved sequence element and stem loop 3 (SL3).

Main Results:

U-CAN-seq successfully detected a competitive disadvantage associated with the N24A mutation in CHIKV.
Mutations disrupting the sequence but not the structure of the CHIKV 5' conserved sequence element did not alter viral fitness.
Mutations in the adjacent CHIKV stem loop 3 (SL3) caused a significant fitness disadvantage compared to wild-type CHIKV.

Conclusions:

U-CAN-seq is a sensitive, cost-effective, and rapid method for assessing RNA virus evolutionary fitness.
Primary sequence determinants within the CHIKV SL3 region play a critical role in viral fitness.
The U-CAN-seq assay provides valuable insights into RNA virus evolution and biology.