The other deadly virus: tiled amplicon sequencing and assembly of African swine fever virus

Abstract

African swine fever virus (ASFV) is the causative agent of a deadly, panzootic disease, infecting wild and domesticated suid populations. Contained for a long time to the African continent, an outbreak of a particularly infectious variant in Georgia in 2007 initiated the spread of the virus around the globe, severely impacting pork production and local economies. The virus is highly contagious and has a mortality of up to 100% in domestic pigs. It is critical to track the spread of the virus, detect variants associated with pathology, and implement biosecurity measures in the most effective way to limit its spread. Due to its size and other limitations, the 170-190 kbp large DNA virus genome has not been well sequenced, with fewer than 200 genome sequences available in public repositories. In this talk, we will present an efficient, low-cost method of sequencing ASFV at scale. The method uses tiled PCR amplification of the virus to achieve greater coverage, multiplexability and accuracy on a portable sequencer than is achievable using shotgun sequencing. We will also introduce Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly, which other methods fail on. The ASFV genomes produced using this method are near complete, lacking only parts of the highly repetitive 3’ and 5’ telomeric regions, and have a high level of accuracy. These methods will allow sequencing of ASFV at optimal efficiency and high throughput to monitor and act on the spread of the virus as part of the global panzootic response.

Learning objectives

• To understand the threat posed by the African swine fever virus (ASFV) and how improved sequencing methods can aid with tracking the virus
• To learn the protocol for amplification and sequencing of ASFV from outbreak samples (Genotype II) and identify pitfalls
• To learn how to use the Lilo pipeline for assembling the data
• To look out for adaptations the method to accommodate other ASFV genotypes

Meet the speakers

Amanda Warr is a postdoc at The Roslin Institute within the University of Edinburgh, UK. She holds a M.Sc in Animal Science, B.Sc in Zoology and a Ph.D. in genetics and genomics. Her Ph.D. project focussed on using genomics to investigate reproductive traits in pigs and reassembling the pig genome using long-read sequencing. Whilst Amanda’s work is primarily focussed in bioinformatics, she spends time in the lab using the MinION and PromethION platforms, training other users, and she also founded a Scottish nanopore user group called ION_BRU. The nanopore-related projects and collaborations she is involved in include work in a variety of species on anti-microbial resistance, viral epidemiology, genome assembly in mammals and microbiomes, and diagnostics. Her main projects focus on microbiome sequencing and tracking the spread of African swine fever virus and porcine reproductive and respiratory syndrome virus in the Philippines, all using long-read sequencing. Recently, Amanda was awarded a BBSRC Discovery Fellowship starting in July 2022.

Christine Tait-Burkard is a group leader at The Roslin Institute/University of Edinburgh and an expert on coronaviruses and arteriviruses. She holds a BSc and MSc in Biology, Major Biochemistry, from the Swiss Federal Institute of Technology, ETH, Zurich, Switzerland and a PhD in Virology from Utrecht University, the Netherlands.
She has worked in some of the world’s leading virology laboratories, obtaining an extensive background in virology and host interactions, with particular expertise in coronavirus and arterivirus research. Dr Tait-Burkard recently established that genome edited pigs with a deletion in part of the PRRSV receptor CD163 are fully refractory to infection with PRRSV (Burkard et al., 2018), and was a finalist for BBSRC Innovator of the Year, 2019. She leads a BBSRC-funded project on “Understanding the CD163 - PRRS virus interaction to improve genetic engineering for resistance” and a Newton-Fund/BBSRC project on “A strategic approach to identifying and combating porcine reproductive and respiratory syndrome virus outbreaks and other porcine viral diseases”. She was recently awarded a BBSRC funded grant “Interferon-stimulated genes as resilience factors for PRRSV infection” (2020-2023). She leads the UoE contribution of SARS-CoV-2 host-pathogen interaction studies for two work packages within the IMI-2 CARE – Corona Accelerated R&D in Europe project (2020-2025) and is involved in several other COVID-associated project. She also leads a number of industry-funded research projects within The Roslin Institute.
Her research focuses on understanding how porcine and zoonotic viruses interact with their hosts and how this understanding can be used to identify genetic susceptibility/resistance and develop diagnostics and treatments.