Validation of an unbiased metagenomic detection assay for RNA viruses in viral transport media and plasma

Publication date: Mar 26, 2024

Unbiased long read sequencing holds enormous potential for the detection of pathogen sequences in clinical samples. However, the untargeted nature of these methods precludes conventional PCR approaches, and the metagenomic content of each sample increases the challenge of bioinformatic analysis. Here, we evaluate a previously described novel workflow for unbiased RNA virus sequence identification in a series of contrived and real-world samples. The novel multiplex library preparation workflow was developed for the Oxford Nanopore Technologies (ONT) MinION sequencer using reverse transcription, whole genome amplification, and ONT’s Ligation Sequencing Kit with Native Barcode Expansion. The workflow includes spiked MS2 Phage as an internal positive control and generates an 8-plex library with 6 samples, a negative control and a gfp transcript positive control. Targeted and untargeted data analysis was performed using the EPI2ME Labs framework and open access tools that are readily accessible to most clinical laboratories. Contrived samples composed of common respiratory pathogens (Influenza A, Respiratory Syncytial Virus and Human Coronavirus 229E) in viral transport media (VTM) and bloodborne pathogens (Zika Virus, Hepatitis A Virus, Yellow Fever Virus and Chikungunya Virus) in human plasma were used to establish the limits of detection for this assay. We also evaluated the diagnostic accuracy of the assay using remnant clinical samples and found that it showed 100% specificity and 62.9% clinical sensitivity. More studies are needed to further evaluate pathogen detection and better position thresholds for detection and non-detection in various clinical sample metagenomic mixtures.

Concepts Keywords
Alphainfluenzaviruses Assay
Biofire Barcode
June Clinical
Library Contrived
Unbiased Control


Type Source Name
disease IDO assay
disease VO Viruses
disease IDO pathogen
pathway KEGG Influenza A
disease VO Respiratory syncytial virus
disease VO Yellow fever virus
disease VO Chikungunya virus
drug DRUGBANK Tropicamide
drug DRUGBANK Coenzyme M
disease MESH Influenza
disease MESH Hepatitis
disease IDO blood
disease MESH COVID 19
disease VO Optaflu
disease VO Metapneumovirus
disease MESH mosquito borne diseases
disease MESH complications
disease MESH viral infections
disease VO effective
disease VO device
drug DRUGBANK Edetic Acid
drug DRUGBANK Phosphate ion
drug DRUGBANK Aspartame
disease IDO host
disease VO viable
disease VO Bacteria
disease IDO organism
disease MESH Parainfluenza
disease IDO process

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