Simultaneous rapid sequencing of multiple RNA virus genomes

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Studying viral evolution and designing vaccines requires analyzing archived viral sequences.
• Generating individual full-length viral sequences is time-consuming, costly, and difficult.
• Next-generation sequencing (NGS) offers potential for pathogen discovery and viral evolution studies.

Purpose of the Study:

• To develop a rapid, cost-effective method for simultaneously sequencing multiple archived viral samples.
• To enable efficient analysis of viral evolution and support vaccine design.
• To overcome the limitations of individual sample sequencing.

Main Methods:

• A novel sequencing procedure using random-primed cDNA synthesis with barcoded primers.
• Nuclease treatment to reduce host nucleic acids, followed by RNA extraction.
• Single-tube cDNA synthesis, PCR amplification, pooling, size fractionation, and sequencing on the Ion Torrent PGM platform.
• Utilized de novo and template-assisted assembly for genome reconstruction.

Main Results:

• Successfully sequenced multiple archived viral samples (bovine pestivirus and coronavirus isolates) simultaneously and rapidly.
• Consistently obtained near full-length or full-length viral genomic sequences.
• The barcoded primers allowed for differentiation of individual viral libraries after pooling and sequencing.

Conclusions:

• The developed procedure is efficient for simultaneous, rapid sequencing of multiple archived viral samples.
• This method facilitates the study of viral evolution and aids in the design of new vaccines.
• The technique overcomes previous limitations in sequencing archived viral isolates, enabling broader genomic analysis.