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.

The authors have declared no competing interest.

This research was funded by Centers for Disease Control and Prevention under award numbers 75D30121C12250 and 75D30122C15359. The views and conclusions contained herein are those of the authors and should not be interpreted as official policy or endorsements of the HHS, CDC, or the US Government.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Human Protections Administrator (HPA) for Signature Science, LLC, as designated by the Federalwide Assurance (FWA) issued by the US Department of Health and Human Services, reviewed the certifications of Institutional Review Board (IRB) review and approval associated with the discarded, deidentified, commercially available human swab samples obtained from two vendors Discovery Life Sciences, BioIVT, and Precision BioSciences that were used in this research study. The Signature Science, LLC HPA found that these three vendors appropriately obtained and documented their IRB approval and donor consent for the collection of the human samples that were then obtained and used in this research project.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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All data produced in the present study are available upon reasonable request to the authors. Metagenomic reads from samples from this study will be depleted of human host sequences and will be submitted to the NCBI BioProject database before peer reviewed publication.