Improvements in metagenomic virus detection by simple pretreatment methods

ABSTRACT

Highlights • Methods assessed for non-viral nucleic acid depletion from clinical samples.• SARS-CoV-2, human papillomavirus (HPV) and molluscum contagiosum virus (MCV) detection used as test of concept.• DNase I treatment followed by filtration dramatically improved virus detection efficiency.• Presenting a metagenomic workflow with Nanopore sequencing for prompt RNA and DNA virus detection.• The Delta variant of SARS-CoV-2, HPV-genotype and a co-infection of HPV and MCV-1 were correctly identified with this workflow. Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified;this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses. This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.