Unbiased long read sequencing approaches for clinical metagenomic sample analysis holds enormous potential for pathogen detection, including improved detection of unknown, novel or emerging viruses. However, the rapid rate of development in nanopore sequencing and library preparation methods complicates the process of selecting a standardized method for unbiased RNA virus detection. Here, we evaluate multiple sequencing approaches to identify a workflow with sufficient sensitivity, limits of detection, and throughput for potential utilization in a clinical laboratory setting. Four separate library preparation methods for the Oxford Nanopore Technologies MinION sequencer are compared, including direct RNA, direct cDNA, rapid cDNA, and double stranded cDNA. We also establish that depletion of host RNA is not required and can be deleterious for viral RNA detection in some instances when using samples in viral transport media (VTM) or plasma. Using unbiased whole genome amplification following reverse transcription, we achieve limits of detection on the order of 1.95E03 GE/mL of Venezuelan Equine Encephalitis Virus (VEEV) spiked in human plasma. We also report initial detection of 5.43E06 GE/mL of coronavirus 229E spiked into VTM samples containing human background RNA which are expected to decrease significantly during upcoming testing. These metrics were achieved within a 6-plex multiplex reaction, illustrating the potential to increase throughput and decrease costs for relevant sample analysis. Data analysis was performed using EPI2ME Labs framework and open access tools that are readily accessible to most clinical laboratories. Taken together, this work describes an optimized method for unbiased nanopore sequencing and analysis of RNA viruses present in two common clinical matrices.

The authors have declared no competing interest.

This research was funded by Centers for Disease Control and Prevention under award numbers 75D30121C12250. 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.

Yes

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.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

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).

Yes

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

Yes

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