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Updated: Aug 7, 2025

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Published on: July 2, 2016

Rapid whole genome sequencing methods for RNA viruses.

Masayasu Misu^1,2, Tomoki Yoshikawa¹, Satoko Sugimoto¹

¹Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.

Frontiers in Microbiology

|March 13, 2023

Summary

New PCR-NGS and RCA-NGS methods enable accurate, economical whole RNA viral genome sequencing without complex sample preparation. These techniques overcome limitations of traditional next-generation sequencing (NGS) for diverse RNA viruses.

Keywords:

MinION nanopore device RNA virus next-generation sequencing – NGS rapid amplification of cDNA ends (RACE)rolling circle amplification (RCA)whole genome sequencing (WGS)

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Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Published on: June 3, 2019

Area of Science:

Virology
Genomics
Molecular Biology

Background:

RNA viruses cause numerous infectious diseases.
Accurate whole RNA viral genome sequencing is crucial but challenging due to complex sample preparation and limitations in determining terminal sequences with conventional Next-Generation Sequencing (NGS).
Existing methods often require nucleic acid enrichment and rapid amplification of cDNA ends (RACE), which is burdensome for segmented genomes.

Purpose of the Study:

To develop novel, efficient, and accurate methods for whole RNA viral genome sequencing.
To overcome the limitations of current NGS techniques, including complex sample preparation and inability to determine terminal sequences.
To establish a method applicable to various RNA virus types, including those with segmented genomes.

Main Methods:

Established PCR-NGS and RCA-NGS methods optimized for the MinION NGS platform.
Implemented a workflow involving nuclease treatment to remove non-viral nucleic acids.
Utilized barcoded linkers for terminal sequence determination and subsequent amplification via linker-specific PCR or rolling circle amplification (RCA).

Main Results:

Achieved 100% accuracy in determining whole RNA viral genome sequences for diverse virus types (single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented, and multi-segmented).
Obtained high mean read depths exceeding 2,500× for all tested viruses.
Demonstrated that the methods do not require virus-specific primers, physical viral particle enrichment, or RACE.

Conclusions:

PCR-NGS and RCA-NGS provide a streamlined, accurate, and economical approach for whole RNA viral genome sequencing.
These novel methods simplify the process and improve accuracy, making them suitable for a wide range of RNA viruses.
The developed techniques facilitate easier and more cost-effective determination of accurate RNA viral genomes.