UBC Theses and Dissertations
Discovery and surveillance of viral spillover threats using probe capture-based targeted genomic sequencing Kuchinski, Kevin Stephen
Human health and agriculture are constantly threatened by viruses that spillover from wildlife. Genomic sequencing is becoming integral to protecting humanity from these threats. This technology is being used to explore viral biodiversity and identify new potential pathogens. It is also being used to monitor human, livestock, and wildlife populations for potential spillovers. Recovering and analyzing viral genomes in real-world specimens remains a challenge, however. Viral genomic material must be recovered from human, animal, and environmental specimens where it is extremely dilute, fragmented, or incomplete. This makes enrichment of viral genomic material necessary for practical, high-throughput surveillance operations. Hybridization probe capture is a powerful technique used in many fields to enrich target genomic material when preparing specimens for sequencing. This technique uses synthetic nucleotide oligomers (probes) to capture target genomic fragments while background material is washed away. To achieve this, collections of probes must be designed with sequences complementary to the target genome, which becomes problematic for viral targets with extensive genetic diversity and hypervariability. Designing compact probe panels that provide broad coverage of viral taxa presents a substantial computational challenge. A lack of accessible probe design tools has kept this powerful technique out of reach for many public health and agriculture agencies. Consequently, probe capture has not been widely deployed for large-scale discovery and routine surveillance of viral spillover threats. To address this gap, this dissertation presents ProbeTools, a user-friendly software package developed to facilitate probe panel design for diverse and hypervariable viral taxa. In this dissertation, ProbeTools is validated in silico on tens of thousands of reference genome sequences and in vitro on dozens of egg-cultured viral isolates. Next, ProbeTools is applied to two real-world viral discovery and surveillance applications. First, it is used to characterize genomes of novel coronaviruses in bats. Second, it is used to detect avian influenza viruses in environmental specimens from wild bird habitats. The validations and case studies in this dissertation demonstrate the power of probe capture for discovery and surveillance of viral spillover threats. They also demonstrate the suitability and flexibility of ProbeTools when designing probe panels for applied viral genomics.
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