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

## TL;DR

ViBE-Seq is a new method for accurately sequencing the 5′ ends of RNA viruses like Ebola and Marburg, using very small RNA samples.

## Contribution

ViBE-Seq introduces a novel, high-fidelity method for sequencing viral RNA ends with minimal input RNA.

## Key findings

- ViBE-Seq reliably sequences the 5′ end of filovirus RNA from virions, infected cells, and animal tissue.
- The method requires as little as 50 ng of total RNA for high-resolution sequencing.
- ViBE-Seq can assess terminal deoxynucleotidyl transferase activity in reverse transcriptases.

## Abstract

Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5′ and 3′ ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5′ end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5′ end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.