Low-Input, High-Resolution 5' Terminal Filovirus RNA Sequencing with ViBE-Seq

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Next-generation sequencing (NGS) often provides insufficient coverage of viral genome 5' and 3' ends.
• Terminal sequences are critical for RNA virus transcription and replication.
• Lack of terminal sequence data impedes the study of emerging and re-emerging viruses.

Purpose of the Study:

• To develop a novel, high-resolution method for sequencing the 5' and 3' ends of viral RNA genomes.
• To overcome limitations in current sequencing techniques for viral genome termini.
• To facilitate comprehensive genomic analysis of emerging RNA viruses.

Main Methods:

• Development of ViBE-Seq (Viral Bona Fide End Sequencing) protocol.
• Application of ViBE-Seq to sequence 5' ends of viral RNA genomes and mRNAs.
• Utilized Ebola virus and Marburg virus as model systems.
• Tested ViBE-Seq with various RNA sources: virions, infected cells, and animal tissues.

Main Results:

• ViBE-Seq achieves high-resolution sequencing of filoviral genome ends with high fidelity.
• The method requires as little as 50 ng of total RNA for 5' end sequencing.
• Demonstrated reliability and accuracy for sequencing RNA from diverse sources.
• ViBE-Seq can identify terminal deoxynucleotidyl transferase activity in reverse transcriptases.

Conclusions:

• ViBE-Seq is a robust and reliable technique for accurate 5' end sequencing of filovirus RNA.
• This method enables access to complete viral genome sequences, essential for studying replication and transcription.
• ViBE-Seq significantly advances the study of emerging and re-emerging RNA viruses.