NGS

Development of a procedure for the diagnosis of outbreaks of emerging, mutated or novel viral pathogens (2016 - ongoing)

Doctoral Thesis of Jakub Kubacki med.vet.

Due to globalization and environmental changes, human and animal health is challenged by an increasing number of viral diseases often caused by emerging or re-emerging viruses and novel viral variants. Classical diagnostic methods are limited to circumscribed groups of known pathogens and require careful triage and a targeted approach. Using next generation sequencing (NGS) technology, identification and discovery of viruses is possible without the need to target a specific infectious agent – all viruses present in a sample are sequenced. Hence, it is ideal for the diagnosis of unexpected or novel viruses. In the wake of decreasing costs for sequencing, NGS is becoming progressively exploitable in the veterinary diagnostic field, particularly in cases of disease outbreaks where standard routine diagnostics such as ELISA or PCR fail to identify the infective agent.

In order to establish a NGS protocol applicable in veterinary diagnostics, several points have to be attained. The protocol should be sensitive, fast, cost-efficient and adaptable to diverse samples materials and species. In a first step, we focused on the sample preparation. Parameters such as filter pore size, nuclease treatment, extraction method and amplification cycle numbers were tested and compared using samples spiked with RNA and DNA viruses.  Effectiveness of each method in order to remove host nucleic acid and preserve viral genomes was measured by specific real-time (RT)-PCR and DNA concentration measurements on Qubit Fluorometer.

Finally, three types of samples from pigs (lung tissue, feces and nasal swabs) were spiked with several known RNA and DNA viruses and divided into four groups depending on the preparation method - from homogenization to the DNA ready for library preparation - and subsequently sequenced in NextSeq Series Desktop Sequencing System at the Functional Genomics Center Zurich (FGCZ). Additionally, three known virus positive tissue and fecal samples and three unknown samples from clinical cases were sequenced. Analysis of the sequence data showed that enrichment for virus particles such as filtration, centrifugation and nuclease treatment as well as amplification have significant influence on the number of viral genomic sequences detected. Based on these results, further steps and benchmarks of the protocol can be tackled. 

Cover Illustration: Alignment of NGS sequencing reads showing an area with a point mutation