Efficient and unbiased metagenomic recovery of RNA virus genomes from human plasma samples

Abstract

RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses – HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at very low levels. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6, and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.

Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples

“…Increasing the proportion of virus RNA for sequencing using cell culture is often difficult and may result in genetic variation that is not representative of the natural sample. Therefore, using total RNA extracted directly from natural host tissue is preferable in order to obtain evolutionarily relevant information on the virus genomes [11].…”

Mosquito-Borne Viral Diseases: Control and Prevention in the Genomics Era

“…Viral discovery and identification from isolates and metagenomic samples present major challenges to bioinformatics in general. This is because viral genomes are prone to very high variability and deviation from reference genomes [42], continuous emergence of new viruses with no available references, high intrapopulation diversity, and the relative rareness of viral DNA fragments in metagenomic samples [43]. These challenges have largely been addressed through the following pipelines.…”

Enhanced Detection of DNA Viruses in the Cerebrospinal Fluid of Encephalitis Patients Using Metagenomic Next-Generation Sequencing

“…Improving this ratio is key to achieving a sufficient amount of viral reads to allow reliable detection and accurate identification of viruses in a sample ( Chan et al, 2014 ; Hall et al, 2014 ; Kohl et al, 2015 ; Lewandowska et al, 2015 ; Bukowska-Ośko et al, 2017 ). Selective depletion of the ribosomal RNA (rRNA) fraction followed by DNAse digestion resulted in a significant methodological improvement in the mNGS protocol for RNA viruses previously developed in our laboratory ( Manso et al, 2017 ). However, effective enrichment of viral DNA has proven to be more challenging due to the lack of differential motifs between human and viral DNA that allow depleting the former without affecting the number of copies of the latter in the sample.…”