Amplification of Filovirus Genomes from Clinical Samples for Next Generation Sequencing

Viral genome sequencing has become a critical tool in outbreak mitigation. Due to their small size relative to the host genome, viral genomes comprise a small fraction of next generation sequencing reads in clinical samples when using unbiased sequencing approaches. Long-range polymerase chain reaction facilitates the amplification of viral genomes from clinical and environmental samples with minimal primer sites, allowing researchers to target regions of the genome that are conserved across available variants. Here, we describe the amplification and sequencing of the Ebola virus genome from tissue samples collected from infected nonhuman primates. This protocol facilitates full viral genome recovery from as low as 10³ median tissue culture infectious doses per milliliter.