Improvement of field-deployable metagenomic virus detection by a simple pretreatment method (preprint)

ABSTRACT

As both the current COVID-19 pandemic and earlier pandemics have shown, animals are the source for some of the deadliest viral pathogens, which can spread to humans. Therefore, early detection at the point of incidence is crucial to both prevent and understand the threats posed to human health from pathogens in animal reservoirs. Often, the exact genetic nature of these zoonotic pathogens is unknown and advanced laboratory facilities do not exist in most field settings and therefore the development of methods for unbiased metagenomic and point of incidence detection is crucial in order to identify novel viral pathogens in animals with zoonotic and pandemic potential. Here we addressed some of these issues by developing a metagenomic Nanopore next-generation sequencing (mNGS) method for nucleic acids extracted from clinical samples from patients with SARS-CoV-2. To reduce the non-RNA viral genetic components in the samples, we used DNase pretreatment in a syringe followed by filtration and found that these pretreatments increased the number of SARS-CoV2 reads by > 500-fold compared with no pretreatment. The simple protocol, described here, allows for fast (within 6 hours) metagenomic detection of RNA viruses in biological samples exemplified by SARS-CoV-2 detection in clinical throat swabs. This method could also be applied in field settings for point of incidence detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory. Highlights Here we present a field-deployable metagenomic Nanopore protocol for RNA virus detection. SARS-CoV-2 detection used as proof-of-concept. Analysis of simple methods for non-viral nucleic acid depletion were tested on SARS-CoV-2 clinical samples. DNase I treatment followed by 0.22µM syringe filtration dramatically increased the number of SARS-CoV-2 reads and virus genome coverage.