Clinical metagenomics for detection of viruses using short-read, long-read and targeted approaches

Background Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, barriers to broader take-up include the need for high sequencing depths, long turnaround times, and limited sensitivity. Newer metagenomics protocols based on Oxford Nanopore Technologies (ONT) sequencing allow acquisition and analysis of data in real time, potentially reducing the need for high-volume sequencing and enabling point-of-care testing. Furthermore, targeted approaches that selectively amplify known pathogens could improve sensitivity. Methods We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Biosciences Viral Research Panel (VRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers. Results Capture with the Twist VRP increased sensitivity by at least 10-100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600-6,000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the VRP and ONT. Conclusions Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist VRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement SB, NA and OETM are funded by the NIHR Blood and Transplant Research Unit in Genomics to Enhance Microbiology Screening. LF and LMMB are funded by the NIHR Great Ormond Street Hospital Biomedical Research Centre. JB receives funding from the NIHR UCL/UCLH Biomedical Research Centre. All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. ### Author Declarations 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 study used only commercially available human genetic material. The products used were Human genomic DNA - G1471 | Promega https://www.promega.co.uk/products/biochemicals-and-labware/nucleic-acids/human-genomic-dna/?catNum=G1521 and Human Brain Total RNA - AM7962 | Invitrogen https://www.thermofisher.com/order/catalog/product/AM7962 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.